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Spermatogenesis in the hologonic testis of the trichuroid nematode, Capillaria hepatica (Bancroft, 1893)
Printed in Sweden Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved
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J. ULTRASTRUCTURE RESEARCH
44, 210-234 (1973)
Spermatogenesis in the Hologonic Testis of the Trichuroid Nematode, Capillaria hepatica (Bancroft, 1893) B. W. NEILL and K. A. WRIGHT1
Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Ontario, Canada Received February 27, 1973 Deve/opment of spermatozoa in the hologonic testis of Capillaria hepatica (Bancroft, 1893) has been examined by light and electron microscopy. The testis contains a region of developing germ cells that may encircle the testis, or b e restricted to a circumferential zone. This germinal region extends the entire length of the testis. Spermatogonia occur closest to the periphery. Spindle remnants are characteristic of division stages of these cells. Primary spermatocytes are identified by the breakdown of the nuclear envelope and expansion of the endoplasmic reticulum. Nuclear material becomes ringed by mitochondria. Karyokinesis is followed by incomplete cytokinesis. The second meiotic division follows rapidly, giving four spermatids that remain connected via cytoplasmic bridges. The cell membrane of both primary and secondary spermatocytes is elaborately folded. During the second meiotic division, superfluous cytoplasm, containing Golgi apparatus and endoplasmic reticulum, is extruded as a residual body. After separation of spermatids, spermateleosis begins. Large diameter tubules occur in the cytoplasm of early spermatids. These probably collapse to form individual membrane loops which coalesce to form a system of interconnected membrane specializations of the developing sperm. Nuclear material progressively condenses to form the dense, nonmembrane-bound nucleus. Spermateleosis continues in the luminal portion of the testis. Sperm develop a head region including the trilobed nucleus, some mitochondria and fl-glycogen, and a tail containing closely packed peripheral membrane specializations and centrally disposed mitochondria. Pseudopodla occur on sperm in the vas deferens and probably in the testis. Throughout reduction division stages, germ cells are enclosed by processes of sustentacular cells. These cells may have three functions: nutritive, regulatory and supportive. The first may be evidenced by phagocytic activity and the elaborations of adjacent spermatocyte membranes. Regulation of sperm development is not a striking feature but may be a function of sustentacular cells 1 Support of Grant A 3757 from the National Research Council of Canada is acknowledged.
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along with cytoplasmic bridges between spermatocytes. Sustentacular cells probably hold early developmental stages in the testis against the flow of mature sperm through the lumen. Nematodes have two modes of germ cell production: telogonic and hologonic (5). In the telogonic testes of Parascaris equorum (=Ascaris rnegalocephala), germ cells are proliferated from the blind proximal end, or germinal zone, apparently from a single terminal cell. The germinal zone is succeeded by a growth zone in which spermatogonia enlarge and differentiate. Further distally is the zone of maturation where spermatids transform into spermatozoa (spermateleosis). Chitwood and Chitwood (5) and Hyman (20) considered this pattern to be representative of telogonic testes in general. In the hologonic testis, proliferation of germ cells takes place along the entire length of the gonad, either around its perimeter, as observed by Leuckart (26) for Dioctophyma renale, or in limited areas of the testis, as observed by Eberth (10) for Trichuris triehiura. Spermateleosis then occurs toward the lumen of the testis. Investigations of spermatogenesis were initiated by Van Beneden and Julin (41) in the telogonic testes of Parascaris equorum and continued by many workers, notably Faur6-Fremiet (11), Sturdivant (40), Pasteels (34), and Favard (12). Germ cell development in other nematode species has received little attention. Cobb (7, 8) described spermatogenesis of Spirina parasitifera a free-living marine nematode2 Although four spermatids resulted initially from two meiotic divisions of the primary spermatocyte, he reported an intercalation of a number of mitotic divisions of the spermatid to form a 128-cell "spermatophore." Re-examination of this species by Chitwood and Chitwood (5) revealed that what Cobb had interpreted as the spermatid was the enormous, hollow spermatozoon. The structures that he interpreted as sPerm: atophore nuclei proved to be rows of secretion globules in the wall of the vas deferens. Four spermatids resulting from two maturation divisions of the primary spermatocyte, subsequently develop into four spermatozoa. Thus, the generalized sequence of nematode spermatogenesis as presented by Walton (44), which was based principally on studies of P. equorum, holds for S. parasitifera. This spermatogenic pattern was also upheld in recent studies on Porrocaecum angustieolle (31) and on the filarids Onchocerca volvulus and Wuchereria bancrofti (29.) Favard (12) traced spermatogenesis in Parascaris equorum with electron microscopy, while aspects of spermatogenesis were examined in Nippostrongylus brasiliensis, Aspiculuris tetraptera, and Rhabditis
pellio (3, 21, 25). Chitwood and Chitwood (5) and Nigon (33) have stated that germ cell production in the hologonic testis of nematodes is similar to that in the seminiferous tubules of mammals. Rauther (36) noted that the testis of Trichuris suis (= Trichocephahts crenatus) was enveloped by a basal lamina and that germ cells were proliferated 15-- 731828 J-. Ultrastructure Research
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toward the lumen from circumferentially located spermatogonial cells, which divided to produce spermatids. He also reported nongerminal features of the testis. The cells which comprised the epithelium in this hologonic gonad apparently did not form a continuous layer, as in telogonic gonads but rather, were scattered throughout the testis. The nuclei of these cells were prominent while the cytoplasmic boundaries were ill-defined. Rauther further observed that "stroma" permeated the interstices between developing germ cells. It is evident that our major understanding of the process of spermatogenesis in nematodes is based on a limited number of studies concerning telogonic testes, especially of Parascaris equorum. It was the aim of this study to determine the sequence of spermatogenesis in the hologonic testis of the nematode Capillaria hepatica (Bancroft, 1893), Travassos, 1915, and to describe the mature sperm. This species is a paraSite of the liver of a wide range of mammalian hosts, and can be maintained in the laboratory in mice. MATERIALS AND METHODS
CapiIlaria hepatica was maintained in the laboratory in mice, using the same methods as in earlier studies (46). Adult nematodes were collected from 18-35 days after infection. Living sperm were dissected from the vas deferens of worms into Tyrode's solution and examined with phase-contrast microscopy. Some sperm were smeared onto slides, fixed in methanol, and stained with Giemsa stain. For light microscopy, pieces of infected liver were fixed in triethanolamine-formalin (TAF) (19), Bouin's, or calcium formal (27). Sections were stained with Harris' hematoxylin and eosin, trichrome, or with the Feulgen reaction for DNA. Portions of worms prepared for electron microscopy were fixed in 5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.7) at 4°C for varying times and post fixed in 1 or 2% osmium in cacodylate for about 2 hr at 4°C. Some tissues were also fixed directly in 2 % osmium in cacodylate (pH 7.7) containing 5% sucrose. All tissue was embedded in an Epon 812-Araldite 502 mixture. Sections were stained with a sequence of potassium permanganate and lead citrate (47) or with 25 % saturated methanolic uranyl acetate and lead citrate. Glycogen was stained by Thi6ry's modified PAS technique (47). RESULTS Observations from light microscopy of sections of paraffin or epoxy-embedded tissues indicate that the cellular organization of the testis is similar throughout its length, with a peripheral region of compact cells, seen by electron microscopy to be FIG. 1. Part of a cross section through the body of a male Capillariahepatica showing three principal regions of the testis: In region A division stages of germ cells occur while inregion B there is a large sustentacular cell. Cytoplasm of the sustentacular cell contains a variety of phagocytic vacuoles and two dense bodies that probably are residual bodies produced during the second meiotic division of spermatogenesis. "Region C represents the lumen region of the testis and contains spermatozoa. Asterisk marks a large cell undergoing degeneration in the testis lumen. BW=body wall, sn= sustentacular cell nucleus?Arrows indicate pseudocoelom, x 5 700.
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developing stages of spermatocytes, and a central region that may be considered a lumen and contains spermatids and spermatozoa (Fig. 1), The only variation evident is that in some areas the peripheral spermatogenic region occurs around the entire circumference, while in others it is limited to one side of the testis. At the light microscope level, a separate cellular wall is not evident. However, at the electron microscope level, the testis is found to be bounded by two basal laminae (Figs. 2 and 20). In places, small muscle cells occur between these laminae (Figs. 2 and 20). Two principal kinds of cells can be distinguished in the testis, spermatogenic cells and sustentacular cells (Figs. 1 and 12). The latter are closely applied to developing sperm up to the early spermatid stage. Spermatogenesis is apparently a continuous process during the life of the adult nematode that extends between 18 and 35 or 40 days after infection (46).
Spermatogonia Spermatogonia are approximately 5 x 3/~m in size, and are located peripherally in the testis, either lying directly on the basal lamina (Fig. 2) or just in from it, surrounded by processes of sustentacular cells. The nucleus of spermatogonia is large with a prominent nucleolus. The cytoplasm contains mitochondria, free ribosomes, a few microtubules, lipid droplets, Golgi apparatus, and endoplasmic reticulum membranes with few if any ribosomes. Spermatogonia divide by mitotic divisions forming daughter cells transiently connected by a spindle bridge about 1/~m in diameter that contains a spindle remnant (Fig. 3). The spindle remnant is comprised of spindle tubules with dense material surrounding them at the middle of the bridge (Fig. 4). The plasma membrane of the bridge is thickened with dense material on its inner surface. Processes of sustentacular cells are closely applied to the bridge. Nuclei and chromosomes are too small to identify chromosome configurations at the light microscope level. Daughter spermatogonia each contain a single centriole surrounded by particles of intermediate density (Fig. 3).
Primary spermatocyte Subsequent to mitotic division, spermatogonia enlarge slightly so that the ratio of cytoplasmic to nuclear volume increases and allows distinction of the primary FIG. 2. A spermatogonial cell at the periphery of the testis. The high ratio of nucleus to cytoplasm is evident. The periphery of the testis is bounded by two thin basal laminas (arrows) and a thin muscle cell (M). The vas deferens (vd) lies close to the testis, x 23 000. FIG. 3. Daughter spermatogonia after mitotic division. Note interphase nuclei (n) in each cell and location of centrioles (arrows). The cells are connected by a spindle bridge. Tissue above the spermatogonia belongs to a sustentacular cell and includes heterogeneous lipid inclusions and a clear vacuole, x 14 500. FIG. 4. Higher magnification of a spindle bridge connecting spermatogonia. Some dense material can be seen between the spindle fibres, x 29 600.
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spermatocyte. Polyribosomes and lipid droplets occur in the cytoplasm along with Golgi apparatus, and some endoplasmic reticulum. The nucleolus appears to be indented by a spherical mass of intermediate density resembling a chromoplast, but no synaptonemal complexes were observed (Fig. 5). Development proceeds with widening of the perinuclear cisterna from about 400 • to 2 000-2 400 ~ (Fig. 7). Areas where the cisterna remains at the 400 ,~ width are marked by accumulations of dense material on both its nucleoplasmic and cytoplasmic sides. Continuity of the outer nuclear membrane with the endoplasmic reticulum occurs and cytoplasmic cisternae of endoplasmic reticulum also expand from 380-400 A to about 800-1 200 A. Polyribosomes appear to increase in number, the Golgi apparatus, mitochondria, and lipid droplets persist. No nucleolus was seen at this stage. The peripheral plasma membrane of the primary spermatocyte develops evaginations, or outfoldings. These outfoldings may be simple or multiple ultimtely forming characteristic membrane stacks (Figs. 7, 9, and 11). After breakdown of the nuclear envelope, nuclear material occurs as areas of 10w granular density from which endoplasmic reticulum, mitochondria, and other cytoplasmic components are excluded (Fig. 9). Golgi apparatus appear to increase in numbers but lipid droplets are absent. Mitochondria, initially dispersed in the cell, subsequently surround the nuclear material (Fig. 10). Spindle fibers appear in the cytoplasm indicating the first meiotic division. A polar view of metaphase I, showing the central centriole and three areas of nuclear material, is illustrated in Fig. 11. In Fig. 12 nuclear material, subdivided into clumps, occurs at opposite poles of the elongate cell prior to cytokinesis. Mitochondria regroup around the divided nuclear material. Endoplasmic reticulum cisternae remain expanded and outfolded stacks of peripheral membrane are complex.
Secondary spermatocyte Cytokinesis is incomplete and results in secondary spermatocytes connected by an intercellular bridge (Fig. 14). These intercellular bridges are distinguished from the cytoplasmic bridges of spermatogonia by the lack of dense materials on the cell membrane and absence of a spindle remnant. Processes of sustentacular cells do not penetrate between the dividing cells. Sequential sections indicate that pairs of secondary spermatocytes connected by only one intercellular bridge do occur. FIG. 5. Primary spermatocyte illustrating the large nucleus, nucleolus and chromoplast (arrow). Note homogeneous (neutral) lipid droplets, and scant endoplasmicreticulum. The heterogeneous lipid inclusion(asterisk) occurs in a sustentacularcell. x 24 300. FIG. 6. Part of a spermatid obliquely sectioned, showing mitochondria and the tubular sheath between them and the cell membrane, x 27 200.
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However, more numerous instances of spermatocytes connected by more than one intercellular bridge indicate that the second meiotic division rapidly follows the first. No interphase nuclei occur. Four spermatocytes are thus interconnected by intercellular bridges. A cytoplasmic lobe is pinched off from the group of spermatids close to the points of interconnections via intercellular bridges. This lobe lacks nuclear material but contains Golgi apparatus, endoplasmicreticulum, mitochondria, and vesicles (Fig. 14). It can be considered a residual body of superfluous cytoplasm. Dense bodies appear within the nuclear material first at the end of the first meiotic division (Fig. 12) and increase in numbers through the second division (Fig. 14). • The sequence of development to early spermatids is illustrated in Fig. 16.
Development of spermatids (spermateleosis) Spermatids do not remain connected by intercellular bridges of meiotic divisions, but are enclosed by processes of sustentacular cells until much of the differentiation of the nucleus and organelles is completed. The densities within nuclear areas of spermatocytes appear to be centers of chromatin condensation. They increase in number and size and ultimately coalesce (Fig. 17). The three nuclear regions identified in secondary spermatocytes persist throughout this condensation, so that three contiguous dense spherical masses ultimately form. A nuclear membrane is absenL The trilobed form of the nucleus can be identified by light microscopy of Feulgen or Giemsa-stained sperm (Fig. 18). A pair of centrioles lies close to the nucleus (Fig. 8). The multiple membrane stacks characteristic of the plasmalemma of spermatocytes do not appear after cell division has completed. However, other characteristic membranous organelles develop. As these organelles may be related to membranous bodies characteristic of other nematode sperm they will be referred to by the term introduced by Foor (16), i.e. membrane specializations. Early spermatids are
FIG. 7. Primary spermatocyte undergoing nuclear breakdown. Note expanded form of nuclear envelope and endoplasmic reticulum membranes. Arrows note points where nuclear envelope is not expanded. The cell is enclosed by fine processes of sustentacular cells. At one point the cell membrane of the spermatocyte is outfolded (of). g = Golgi apparatus, x 19 400. FIG. 8. One of a pair of centrioles composed of nine single tutules close to dense nuclear material in a late spermatid. × 63 000. FIG. 9. Primary spermatocyte immediately after breakdown of the nuclear envelope. Nuclear region (n) is of lower granular density, mitochondria are scattered, g = Golgi apparatus, of= outfoldings of cell membrane x 19 700. FIG. 10. Primary spermatocyte in which nuclear meterial (n) is ringed by mitochondria. Note expanded form of endoplasmic reticuluha. The spermatocyte is enclosed by cytoplasm of sustentacular cells that contains mitochondria (arrows) smaller than those of the spermatocyte, x 17 000. FIG. 11. Polar view of a dividing primary spermatocyte illustrating three areas of nuclear material (n) and spindle fibers converging to a point close to a centriole (arrow). Note expanded form of endoplasmic reticulum and outfoldings (of) of cell membrane, g = Golgi apparatus. × 22 300.
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characterized by containing many tubules (Figs. 15 and 17), 850-1 250 • in diameter. Tubules appear to collapse upon themselves forming concave vesicles with an intermembrane space of 260-380 A . These in turn appear to close upon themselves and fuse with adjacent units to give double membrane loops enclosing a portion of cytoplasm. These membrane specializations ultimately come to lie just below the cell membrane around the tail portion of the mature sperm (Figs. 20, 21, and 23).
Spermatozoa As nuclear material condenses to form the trilobed nucleus, the mitochondria remain encircling the nuclear region (Fig. 17). A sheath composed of tubules (each about 180-200 A in diameter) lies between the mitochondria and cell membrane (Figs. 6, 15, and 17). With the complete condensation of the nucleus, the spermatid begins to undergo its final change in form to that of the spermatozoon. This involves elongation of the cell and distribution of the membrane specializations peripherally in one half, arbitrarily referred to as the tail. The tubular sheath breaks down and mitochondria become distributed throughout the sperm. The mature sperm is about 14 × 2 #m and shows a separation by a slight constriction into a blunter head region and a more tapering tail (Fig. 19). The trilobed, Feulgen-positive nucleus lies in the head portion close to the middle of the sperm. Although mitochondria als0 occur, m u c h of the cytoplasm contains fi-glycogen granules. These are stained in electron microscope preparations by the modified PAS reaction and account for PAS-positive, diastase-sensitive staining of the sperm seen in light microscopy. Membrane specializations are closely packed around the periphery of the tail while mitochondria and some fl-glycogen occurs centrally. Within the lumen of the membrane specializations are fine filaments about 100 A thick (Figs. 22 and 23). In late spermatids and spermatozoa the cytoplasm contains an anastam0sing network of material that, especially near the cell periphery, appears tubular (Figs. 20 and 23). This does not clearly resemble agranular endoplasmic reticulum but may be only the fixation pattern of the glycogen-rich cytoplasmic matrix. Pseudopodia projec t irregularly from both head and tail of the sperm but more were found on the head. They were first seen in sperm in the lumen of the testis but occur in greater numbers on sperm in the vas deferens (Fig. 21). In phase-contrast light microscopy they appear as very fine threads. No motility was noted in sperm dissected into Tyrodes' physiological salt solution and examined by phase microscopy. Sperm seen in the uterus of a limited number of female worms resembled those in the vas deferens of males. Figure 24 illustrates stages in transformation of spermatids into spermatozoa. FIG. 12. Section through dividing primary spermatocyte. A sustentacular Cell encloses the spermatocyte, g=Golgi apparatus, n=nuclear material, sn =sustentacular cell nucleus, x 14 000. FIG. 13. A phagocytic vacuole in a sustentacular cell. x 5 600.
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Fro. 16. Diagrammatic summary of spermatid development. Spermatogonia (a) undergo mitotic divisions that form spindle bridges between daughter cells and interphase nuclei reform (b). Primary spermatocytes enlarge; the nuclear envelope and endoplasmic reticulum expands (c). After the nuclear envelope breaks down, chromatin is ringed by mitochondria (d). The first meiotic division is characterized by a cytoplasmic bridge connecting secondary spermatocytes (e). The second meiotic division follows giving a tetrad of spermatids connected by cytoplasmic bridges, and a residual body of cytoplasm is also pinched off (f). Outfoldings of the plasmalemma that develop first on the primary spermatocyte (c-d) and are present through to the tetrad stage (f), have been omitted from the diagram for clarity.
Sustentacular cells S u s t e n t a c u l a r cells envelop s p e r m a t o g e n i c cells up to the early s p e r m a t i d stage. T h e y are recognizeable b y their large p l e o m o r p h i c nuclei (especially w h e n fixed directly in o s m i u m ) a n d extensive c y t o p l a s m (Figs. 1 a n d 12). T h e nucleus has a large nucleolus a n d finely scattered c h r o m a t i n . Centrioles were seen close to the nucleus in a few s u s t e n t a c u l a r cells. E n d o p l a s m i c reticulum a n d G o l g i a p p a r a t u s occur as well as m i t o c h o n d r i a t h a t are smaller t h a n those in germinal cells (Fig. 10). L i p i d inclusions are b o t h in the f o r m of h o m o g e n e o u s d r o p l e t s ( p r o b a b l y n e u t r a l FIo. 14. Division stage of secondary spermatocytes showing spermatids (1-3) connected by cytoplasmic bridges (arrows) and a residual body (RB) containing three Golgi apparatuses (g), mitochondria, and light vacuoles. Small dense bodies occur in the nuclear region, x 13 600. FIG. 15. Spermatids containing large tubules. A dense nuclear body occurs in one spermatid and the sheath between mitochondria and cell membrane is indicated (arrows). Tubules appear to be collapsing to form membrane specializations, x 18 400.
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FIG. 17. Two spermatids showing condensation of nuclear material in one and collapsing tubules in the cytoplasm (arrows) × 31 000.
FI6. 18. Photomicrograph of a sperm stained with Giemsa showing the trilobed nucleus, x 1 360. FIO. 19. Phase-contrast photomicrograph of living sperm showing division into head and tail regions by slight constriction (arrow). x 1 170. 1 ~ . 20. Sperm at the periphery of the lumen of the testis. The tail region contains membrane specializations and mitochondria. Two of the three nuclear lobes are evident. Two basal laminas and a muscle cell form the wall of the testis, p s - p s e u d o c o e l o m , n=nucleus, x35 300.
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FIG. 24. Diagrammatic summary of spermatid differentiation into sperm. Nuclear material is initially dispersed with a few dense bodies within it. Chromatin condensation progresses to form a trilobed, dense, non membrane-bound nucleus in the sperm. Mitochondria are arranged around the nuclear region during this chromatin condensation and a fine tubular sheath lies external to them. The sheath disappears as mitochondria redistribute in the sperm. Irregularly expanded endoplasmic reticulum membranes of early stages may transform into tubular units that progressively collapse upon themselves and unite with neighbors to form membrane specializations in the tail of the sperm. Centrioles remain close to the nucleus. The sperm is separated by a slight constriction into head and tail regions. Pseudopodial projections are most common on the head region in sperm in the vas deferens. Beta glycogen (not shown) occurs in the cytoplasm of the head region and to a lesser extent in the taiI region of the mature sperm.
lipid) a n d m u l t i l a m i n a t e o r w h o r l e d s t r u c t u r e s ( p r o b a b l y p h o s p h o l i p i d ) . W i t h i n t h e m a s s of c o m p a c t cells w h e r e d i f f e r e n t i a t i o n of early s p e r m a t i d s is o c c u r r i n g , sustenta c u l a r cells h a v e fine cell p r o c e s s e s e n c l o s i n g t h e g e r m i n a l cells (Figs. 10, 11, a n d 12). I n s o m e areas, t h e v o l u m e of s u s t e n t a c u l a r cell c y t o p l a s m is larger. H e r e o c c u r e m p t y v a c u o l e s , a n d v a c u o l e s e n c l o s i n g v a r y i n g q u a n t i t i e s of d e n s e g r a n u l a r a n d m e m b r a n o u s m a t e r i a l s (Fig. 13). A l s o , e n c l o s e d b y s u s t e n t a c u l a r cells are large m a s s e s r a n g ing f r o m t h e size of r e s i d u a l b o d i e s of s p e r m a t o c y t e s to t h e size of e n t i r e g o n i a l cells. FIef. 21. Sperm in the vas deferens show numerous irregular pseudopodial projections. Arrow indicates a centriole, n - nuclei, x 25 600. FIG. 22. Section through some of the membrane specializations of the tail of sperm showing crosssectioned filaments (arrows) within them. x 74 200. FIG. 23. Section through two tails and a head region of sperm in the vas deferens showing longitudinal profile of a filament (arrow) within a membrane specialization. × 44 600.
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These have a dense matrix containing mitochondria and other remnants of organelles. They appear to be degenerated germ cells. Some of these occur also in the lumen of the testis among spermatozoa. Here they are not enclosed by sustentacular cells. DISCUSSION The sequence of sperm development in Capillaria hepatica agrees with the general gametogenic pattern of nematodes (44). The cell architecture of spermatogonia and spermatocytes in general agrees with those of similar stages in Parascaris equorum (12) and Rhabditis pellio (3). Spindle bridges formed during division of spermatogonia in C. hepatica resemble spindle bridges characteristic of mitotic divisions in somatic cells (13). In contrast, divisions of spermatocytes are characterized by the formation of cytoplasmic bridges. As spindle remnants are absent from these bridges it is possible that they allow more effective communication between daughter cells and may be important in synchronizing the development of interconnected germ cells (13). In mammals, up to 15 spermatids may be interconnected by intercellular bridges (32). From light microscopic studies of nematode spermatogenesis, interconnection of four spermatids giving rise to tetrad formations seems most common (29, 31, 44). In C. hepatica, four spermatids are briefly interconnected at the end of the second meiotic division. Roosen-Runge (38), Nieander (32), and Courot et al. (9) have suggested that synchrony of sperm development is controlled more through the action of sustentacular cells. In C. hepatica, sustentacular cells are closely applied to spermatogenic cells up to the early spermatid. Spermatogenesis in C. hepatica does not result in the formation of spermatophores. Within the testis, development of sperm does not appear to be highly synchronized but in general does resemble the sequence of differentiation seen in seminiferous tubules of vertebrates. The release of extraneous cytoplasm from nematode spermatocytes as a residual body has been noted previously (2, 3, 7, 8, 12, 17, 22, 29, 31, 40, 41). In C. hepatica, as in vertebrates, release of residual bodies and separation of spermatids occurs simultaneously (14). Spermiation (release of spermatids from sustentacular cells) occurs in mammals by swelling of cisternae of endoplasmic reticulum of the sustentacular cell (42, 43). In this process, spermatids are released while residual bodies remain trapped in the sustentacular cell. Sustentacular cells in the testis of C. hepatica contain materials resembling residual bodies as well as discrete phagocytic vacuoles. This suggests that material of the residual body is degraded and possibly reutilized in the testis. In areas with sustentacular cells and sometimes within the lumen of the testis among sperm, large dense cells occur. These seem to be degenerate germ cells of various stages of development. In the human fetal testis similarly degenerated
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germ cells occur (18), and it is suggested that their phagocytosis by Sertoli cells may contribute to the nutritional requirements of the testis. A similar phenomenon probably occurs in c. hepatica when degeneration initiates in cells associated with sustentacular cells. However, degeneration of later stages may sometimes result in release of these cells along with sperm. Throughout spermatocyte stages, the peripheral cell membrane is elaborately folded. Such folds may be important in uptake of nutrients from sustentacular cells. After separation of spermatids, the outfoldings are absent. Throughout spermatocyte stages in C. hepatica, mitochondria occur around the nuclear region. A similar nuclear-cytoplasmic association was noted for Parascaris equorum (12), Ascaris lumbricoides, Gnathostoma sp., and Dirofilaria immitis (17), and for Rhabditis pelIio (3). In C. hepatica the mitochondria remain closely associated with the nuclear region until condensation of nuclear material is complete, but they then disperse through the sperm. During the period of nuclear condensation the tubular sheath occurs around the mitochondria and may serve to retain the mitochondria in position. A similarly intimate relationship exists between mitochondria and condensing nuclear material in sperm of Hydra littoraIis (45). During the past decade the ultrastructure of sperm from several nematodes has been described (6, 12, 21, 25, 35, 48). Foor (17) characterized four sperm types-ascaroid, strongyloid, diotophymoid, and oxyuroid. As the sperm of C. hepatica does not conform to any of these, a fifth, trichuroid type may be appropriate. The only valid generalizations are that nematode sperm lack flagella, lack an acrosome, and form pseudopodia. The nucleus of sperm of C. hepatica resemble other nematode sperm in lacking a bounding membrane. The trilobed shape of the nucleus reflects the haploid chromosome number. A maximum of three chromatin regions were seen in each pole of dividing spermatocytes. Miller (29) found that chromosomes of Onchocerca volvulus and Wuchereria bancrofti remained distinct throughout spermatogenesis and in the spermatozoa. Centrioles in developing and mature sperm are atypical as they contain only nine single tubules rather than triplets of tubules. Similar centrioles have been found in Nippostrongylus brasiliensis and Ancylostoma duodenale (17, 21). Membrane specializations are characteristic organelles in nematode sperm. Those in C. hepatica do not resemble those of the other sperm types described by Foor (17). The several types of membrane specializations found might reflect differences in their mode formation. It was concluded that membrane specializations of Parascaris equorum, Panagrellus silusiae, and Rhabditis pellio developed from Golgi dictyosomes (3, 12, 35), although Foor (17) suggested that the similar-appearing membrane specializations of Ascaris lumbricoides might develop from mitochondria. The origin
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of the large-diameter tubules from which the membrane specializations differentiate in C. hepatica is not clear. As the irregularly expanded endoplasmic reticulum of earlier spermatocytes is not recognizeable in spermatids, it may have been transformed into the tubules. On the other hand, the membrane specializations of the sperm do resemble membrane loops in the peripheral cytoplasm of tick spermatocytes. These form from invaginations of the peripheral membrane that pinch off to give vesicles (4). Ultimately the membrane loops fuse to form the central vacuole and motile processes of the tick's sperm (37). The membrane specializations of C. hepatica resemble bodies in the sperm of the rotifer Asplanchna sp. (23). The possible role of membrane specializations as acrosomal elements (6, 12) has been eliminated by Foor's observations (16) that the sperm of Ascaris penetrates the oolemma by means of pseudopodia formed on the region of the sperm that lacks membrane specializations. Foor (17) has suggested that final maturation of sperm (capacitation) may occur in utero, and may involve opening of membrane specializations through the surface. Sperm seen around oocytes in females of C. hepatica resembled sperm in the male tract; membrane specializations had not opened to the surface. The identification of sustentacular cells in the testis of C. hepatica confirms Rauther's observations (36) of nongerminal tissue in the hologonic testis, and supports the parallel between these gonads and seminiferous tubules of vertebrates as noted by Chitwood and Chitwood (5) and Nigon (33). Sustentacular cells, known best in vertebrates as Sertoli cells, are referred to as nurse cells, nutritive cells, or support cells in many invertebrates (1, 24, 28, 30, 49). Sato et al. (39) identified supporting cells surrounding spermatogonia in the trematode Paragonimus miyazakii but Featherston (15) did not describe them from the cestode Taenia hydatigena. The role of sustentacular cells is probably 3-fold as in vertebrates--nutritional, regulatory and supportive. The first two of these roles have already been mentioned. The enclosing of spermatocytes by sustentacular cells and the elaborate outfoldings of the spermatocyte membrane may be related to nutrient transfer to the germ cells while phagocytosis of residual bodies and degenerating germ cells may indicate recycling of nutrients. The regulation or synchronization of gamete development may be mediated by either or both sustentacular cells or cytoplasmic bridge connections between spermatocytes. As the testis is a single tubular organ, germinal cells developing along its perimeter would be carried into the flow of sperm that pass through the central lumen unless they were retained in position. This is the supportive role of sustentacular cells. REFERENCES 1. ANDERSON,W. A., and ELLIS, R. A., Z. Zellforsch. Mikrosk. Anat. 77, 80 (1967). 2. ANYA,A. O., Parasitology, 56, 347 (1966). 3. BEAMS,H. W. and SEKHON,S. S., or. Ultrastruct. Res. 38, 511 (1972).
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4. BREUCKER,H., and HORSTMANN,E., Z. Zellforsch. Mikrosk. Anat. 123, 18 (1972). 5. CHITWOOD, B. G. and CmTWOOD, M. B., Introduction to Nematology, Section I. Monumental Printing Co., Baltimore, Md., 1950. 6. CLARK, W. H., MORRETTI,R. L., and T~OMSON, W. W., Exp. Cell Res. 47, 643 (1967). 7. COBB, N. A., J. Hered. 16, 357 (1925). 8. - J. Wash. Acad. Sci. 18, 37 (1928). 9. COUROT, M., HOCHEREAU-DEREVIERS, M. T. and ORTAVANT, R., in JOHNSON, A. D., GOMES, W. R. and VANDEMARK,N. L. (Eds.), Spermatogenesis, Vol. I. Academic Press, New York 1970. 10. EBERTH, C. J., Wiss. Zool. 10, 383 (1860). 11. FAURt~-FR~MIET,E., Arch. Anat. Microsc. Morphol. Exp. t5, 435 (1913). 12. FAVARD, P., Ann. Sci. Nat. Zool. 3, 53 (1961). 13. FAWCETT,D. W., Exp. Cell Res. Suppl. 8, 174 (1961). 14. FAWCETT,D. W., ITO, S. and SLAUTTERBACK,D. B., J. Ceil Biol. 5, 453 (1959). 15. FEATHERSTON,D. W., Z. Parasitenk. 37, 148 (1971). 16. FOOR, W. E., J. Cell Biol. 39, 119 (1968). 17. - Biol. Reprod. Suppl 2, 177 (1970). 18. GONDOS,B. and HOBEL, C. J., Z. Zellforsch. Mikrosk. Anat. 119, 1 (1971). 19. GOODEY,J. B., Laboratory Methods for Work with Plant and Soil Nematodes. H. M. Stationary Office, London, 1957. 20. HYMEN,L. H., The Invertebrates, Vol. III. McGraw-Hill, New York, 1951. 21. JAMUAR, M. P., J. Cell Biol. 31, 381 (1966). 22. KANWAR, U. and ANAND, R., J. Parasitol. 57, 193 (1971). 23. KOErtLER, J. K., Z. Zellforsch. Mikrosk. Anat. 67, 57 (1965). 24. LANGRETH,S. G., J. Cell Biol. 43, 575 (1969). 25. LEE, D. L. and ANYA, A. O., or. Cell Sci. 2, 537 (1967). 26. LEUCKART,R. F., Die Menschlichen Parasiten, Vol. II. Leipzig, 1876. 27. LmLIE, R. D., Histopathologic Technic and Practical Histochemistry. Blackiston, New York, 1954. 28. LONGO, F. J., and ANDERSON, E., J. Ultrastruct. Res. 27, 435 (1969). 29. MILLER, M. J., Can. J. Zool. 44, 1003 (1966). 30. MOSES, M. J., J. Cell Biol. 10, 301 (1961). 31. NATH, V., GUPTA, B. L. and KOCnAR, D. M., or. Microsc. Sci. 102, 39 (1966). 32. NICANDER,L., Z. Zellforsch. Mikrosk. Anat. 83, 375 (1967). 33. NINON, V., in GRASSY,P. P. (ed.), Trait6 de Zoologie, Vol. IV. Masson et Cie, Paris, 1965. 34. PASTEELS,J., Arch. Biol. 59, 405 (1948). 35. PASTERNAK,J. and SAMOILOFF,M. R., Can. J. Zool. 50, 147 (1972). 36. RAUTHER,M., Zool. Jahrb. 40, 441 (1918). 37. Reger, J. F., J. Ultrastruct. Res. 7, 550 (1962). 38. ROOSEN-RUNGE,E. C., Biol. Rev. 37, 343 (1962). 39. SATO, M., Or~, M. and SAKODA,K., Z. Zellforsch. Mikrosk. Anat. 77, 232 (1967). 40. STURDIVANT,H. P., J. Morphol. 55, 435 (1934). 41. VAN BENEDEN, E. and JULIN, C., Bull. Acad. Roy. Belg. 7, 312 (1884). 42. VITALE-CALPE,R., Z. Zellforseh. Mikrosk. Anat. 105, 222 (1970). 43. VITALE-CALPE,R. and BURGOS, M. H., J. Ultrastruct. Res. 31, 381 (1970).
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44. WALTON,A. C., in CHITWOOD,B. G. and CHRISTIE,J. R. (eds.), An Introduction to Nematology, Section II. Leader Press, Babylon, NY, 1940.
45. WE1SSMAN,A., LENTZ,T. L. and BARRNETT,R. J., J. Morphol 128, 229 (1969). 46. WRIGHT,K. A., Can. J. ZooL 38, 167 (1961). 47. WRIGHT,K. A. and DICK, T. A., Z. Parasitenk. 40, 75 (1972). 48. WRIGHT, K. A., HOPE, W. D. and JONES, N. 0., Proc. Helminthol. Soc. Wash. 40, 30 (1973). 49. YASUZUMI,G., TANAKA,H. and TEZUKA,O., J. Cell BioL 7, 499 (1960).
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Spermatogenesis in the Hologonic Testis of the Trichuroid Nematode, Capillaria hepatica (Bancroft, 1893) B. W. NEILL and K. A. WRIGHT1
Department of Parasitology, School of Hygiene, University of Toronto, Toronto, Ontario, Canada Received February 27, 1973 Deve/opment of spermatozoa in the hologonic testis of Capillaria hepatica (Bancroft, 1893) has been examined by light and electron microscopy. The testis contains a region of developing germ cells that may encircle the testis, or b e restricted to a circumferential zone. This germinal region extends the entire length of the testis. Spermatogonia occur closest to the periphery. Spindle remnants are characteristic of division stages of these cells. Primary spermatocytes are identified by the breakdown of the nuclear envelope and expansion of the endoplasmic reticulum. Nuclear material becomes ringed by mitochondria. Karyokinesis is followed by incomplete cytokinesis. The second meiotic division follows rapidly, giving four spermatids that remain connected via cytoplasmic bridges. The cell membrane of both primary and secondary spermatocytes is elaborately folded. During the second meiotic division, superfluous cytoplasm, containing Golgi apparatus and endoplasmic reticulum, is extruded as a residual body. After separation of spermatids, spermateleosis begins. Large diameter tubules occur in the cytoplasm of early spermatids. These probably collapse to form individual membrane loops which coalesce to form a system of interconnected membrane specializations of the developing sperm. Nuclear material progressively condenses to form the dense, nonmembrane-bound nucleus. Spermateleosis continues in the luminal portion of the testis. Sperm develop a head region including the trilobed nucleus, some mitochondria and fl-glycogen, and a tail containing closely packed peripheral membrane specializations and centrally disposed mitochondria. Pseudopodla occur on sperm in the vas deferens and probably in the testis. Throughout reduction division stages, germ cells are enclosed by processes of sustentacular cells. These cells may have three functions: nutritive, regulatory and supportive. The first may be evidenced by phagocytic activity and the elaborations of adjacent spermatocyte membranes. Regulation of sperm development is not a striking feature but may be a function of sustentacular cells 1 Support of Grant A 3757 from the National Research Council of Canada is acknowledged.
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along with cytoplasmic bridges between spermatocytes. Sustentacular cells probably hold early developmental stages in the testis against the flow of mature sperm through the lumen. Nematodes have two modes of germ cell production: telogonic and hologonic (5). In the telogonic testes of Parascaris equorum (=Ascaris rnegalocephala), germ cells are proliferated from the blind proximal end, or germinal zone, apparently from a single terminal cell. The germinal zone is succeeded by a growth zone in which spermatogonia enlarge and differentiate. Further distally is the zone of maturation where spermatids transform into spermatozoa (spermateleosis). Chitwood and Chitwood (5) and Hyman (20) considered this pattern to be representative of telogonic testes in general. In the hologonic testis, proliferation of germ cells takes place along the entire length of the gonad, either around its perimeter, as observed by Leuckart (26) for Dioctophyma renale, or in limited areas of the testis, as observed by Eberth (10) for Trichuris triehiura. Spermateleosis then occurs toward the lumen of the testis. Investigations of spermatogenesis were initiated by Van Beneden and Julin (41) in the telogonic testes of Parascaris equorum and continued by many workers, notably Faur6-Fremiet (11), Sturdivant (40), Pasteels (34), and Favard (12). Germ cell development in other nematode species has received little attention. Cobb (7, 8) described spermatogenesis of Spirina parasitifera a free-living marine nematode2 Although four spermatids resulted initially from two meiotic divisions of the primary spermatocyte, he reported an intercalation of a number of mitotic divisions of the spermatid to form a 128-cell "spermatophore." Re-examination of this species by Chitwood and Chitwood (5) revealed that what Cobb had interpreted as the spermatid was the enormous, hollow spermatozoon. The structures that he interpreted as sPerm: atophore nuclei proved to be rows of secretion globules in the wall of the vas deferens. Four spermatids resulting from two maturation divisions of the primary spermatocyte, subsequently develop into four spermatozoa. Thus, the generalized sequence of nematode spermatogenesis as presented by Walton (44), which was based principally on studies of P. equorum, holds for S. parasitifera. This spermatogenic pattern was also upheld in recent studies on Porrocaecum angustieolle (31) and on the filarids Onchocerca volvulus and Wuchereria bancrofti (29.) Favard (12) traced spermatogenesis in Parascaris equorum with electron microscopy, while aspects of spermatogenesis were examined in Nippostrongylus brasiliensis, Aspiculuris tetraptera, and Rhabditis
pellio (3, 21, 25). Chitwood and Chitwood (5) and Nigon (33) have stated that germ cell production in the hologonic testis of nematodes is similar to that in the seminiferous tubules of mammals. Rauther (36) noted that the testis of Trichuris suis (= Trichocephahts crenatus) was enveloped by a basal lamina and that germ cells were proliferated 15-- 731828 J-. Ultrastructure Research
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toward the lumen from circumferentially located spermatogonial cells, which divided to produce spermatids. He also reported nongerminal features of the testis. The cells which comprised the epithelium in this hologonic gonad apparently did not form a continuous layer, as in telogonic gonads but rather, were scattered throughout the testis. The nuclei of these cells were prominent while the cytoplasmic boundaries were ill-defined. Rauther further observed that "stroma" permeated the interstices between developing germ cells. It is evident that our major understanding of the process of spermatogenesis in nematodes is based on a limited number of studies concerning telogonic testes, especially of Parascaris equorum. It was the aim of this study to determine the sequence of spermatogenesis in the hologonic testis of the nematode Capillaria hepatica (Bancroft, 1893), Travassos, 1915, and to describe the mature sperm. This species is a paraSite of the liver of a wide range of mammalian hosts, and can be maintained in the laboratory in mice. MATERIALS AND METHODS
CapiIlaria hepatica was maintained in the laboratory in mice, using the same methods as in earlier studies (46). Adult nematodes were collected from 18-35 days after infection. Living sperm were dissected from the vas deferens of worms into Tyrode's solution and examined with phase-contrast microscopy. Some sperm were smeared onto slides, fixed in methanol, and stained with Giemsa stain. For light microscopy, pieces of infected liver were fixed in triethanolamine-formalin (TAF) (19), Bouin's, or calcium formal (27). Sections were stained with Harris' hematoxylin and eosin, trichrome, or with the Feulgen reaction for DNA. Portions of worms prepared for electron microscopy were fixed in 5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.7) at 4°C for varying times and post fixed in 1 or 2% osmium in cacodylate for about 2 hr at 4°C. Some tissues were also fixed directly in 2 % osmium in cacodylate (pH 7.7) containing 5% sucrose. All tissue was embedded in an Epon 812-Araldite 502 mixture. Sections were stained with a sequence of potassium permanganate and lead citrate (47) or with 25 % saturated methanolic uranyl acetate and lead citrate. Glycogen was stained by Thi6ry's modified PAS technique (47). RESULTS Observations from light microscopy of sections of paraffin or epoxy-embedded tissues indicate that the cellular organization of the testis is similar throughout its length, with a peripheral region of compact cells, seen by electron microscopy to be FIG. 1. Part of a cross section through the body of a male Capillariahepatica showing three principal regions of the testis: In region A division stages of germ cells occur while inregion B there is a large sustentacular cell. Cytoplasm of the sustentacular cell contains a variety of phagocytic vacuoles and two dense bodies that probably are residual bodies produced during the second meiotic division of spermatogenesis. "Region C represents the lumen region of the testis and contains spermatozoa. Asterisk marks a large cell undergoing degeneration in the testis lumen. BW=body wall, sn= sustentacular cell nucleus?Arrows indicate pseudocoelom, x 5 700.
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developing stages of spermatocytes, and a central region that may be considered a lumen and contains spermatids and spermatozoa (Fig. 1), The only variation evident is that in some areas the peripheral spermatogenic region occurs around the entire circumference, while in others it is limited to one side of the testis. At the light microscope level, a separate cellular wall is not evident. However, at the electron microscope level, the testis is found to be bounded by two basal laminae (Figs. 2 and 20). In places, small muscle cells occur between these laminae (Figs. 2 and 20). Two principal kinds of cells can be distinguished in the testis, spermatogenic cells and sustentacular cells (Figs. 1 and 12). The latter are closely applied to developing sperm up to the early spermatid stage. Spermatogenesis is apparently a continuous process during the life of the adult nematode that extends between 18 and 35 or 40 days after infection (46).
Spermatogonia Spermatogonia are approximately 5 x 3/~m in size, and are located peripherally in the testis, either lying directly on the basal lamina (Fig. 2) or just in from it, surrounded by processes of sustentacular cells. The nucleus of spermatogonia is large with a prominent nucleolus. The cytoplasm contains mitochondria, free ribosomes, a few microtubules, lipid droplets, Golgi apparatus, and endoplasmic reticulum membranes with few if any ribosomes. Spermatogonia divide by mitotic divisions forming daughter cells transiently connected by a spindle bridge about 1/~m in diameter that contains a spindle remnant (Fig. 3). The spindle remnant is comprised of spindle tubules with dense material surrounding them at the middle of the bridge (Fig. 4). The plasma membrane of the bridge is thickened with dense material on its inner surface. Processes of sustentacular cells are closely applied to the bridge. Nuclei and chromosomes are too small to identify chromosome configurations at the light microscope level. Daughter spermatogonia each contain a single centriole surrounded by particles of intermediate density (Fig. 3).
Primary spermatocyte Subsequent to mitotic division, spermatogonia enlarge slightly so that the ratio of cytoplasmic to nuclear volume increases and allows distinction of the primary FIG. 2. A spermatogonial cell at the periphery of the testis. The high ratio of nucleus to cytoplasm is evident. The periphery of the testis is bounded by two thin basal laminas (arrows) and a thin muscle cell (M). The vas deferens (vd) lies close to the testis, x 23 000. FIG. 3. Daughter spermatogonia after mitotic division. Note interphase nuclei (n) in each cell and location of centrioles (arrows). The cells are connected by a spindle bridge. Tissue above the spermatogonia belongs to a sustentacular cell and includes heterogeneous lipid inclusions and a clear vacuole, x 14 500. FIG. 4. Higher magnification of a spindle bridge connecting spermatogonia. Some dense material can be seen between the spindle fibres, x 29 600.
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spermatocyte. Polyribosomes and lipid droplets occur in the cytoplasm along with Golgi apparatus, and some endoplasmic reticulum. The nucleolus appears to be indented by a spherical mass of intermediate density resembling a chromoplast, but no synaptonemal complexes were observed (Fig. 5). Development proceeds with widening of the perinuclear cisterna from about 400 • to 2 000-2 400 ~ (Fig. 7). Areas where the cisterna remains at the 400 ,~ width are marked by accumulations of dense material on both its nucleoplasmic and cytoplasmic sides. Continuity of the outer nuclear membrane with the endoplasmic reticulum occurs and cytoplasmic cisternae of endoplasmic reticulum also expand from 380-400 A to about 800-1 200 A. Polyribosomes appear to increase in number, the Golgi apparatus, mitochondria, and lipid droplets persist. No nucleolus was seen at this stage. The peripheral plasma membrane of the primary spermatocyte develops evaginations, or outfoldings. These outfoldings may be simple or multiple ultimtely forming characteristic membrane stacks (Figs. 7, 9, and 11). After breakdown of the nuclear envelope, nuclear material occurs as areas of 10w granular density from which endoplasmic reticulum, mitochondria, and other cytoplasmic components are excluded (Fig. 9). Golgi apparatus appear to increase in numbers but lipid droplets are absent. Mitochondria, initially dispersed in the cell, subsequently surround the nuclear material (Fig. 10). Spindle fibers appear in the cytoplasm indicating the first meiotic division. A polar view of metaphase I, showing the central centriole and three areas of nuclear material, is illustrated in Fig. 11. In Fig. 12 nuclear material, subdivided into clumps, occurs at opposite poles of the elongate cell prior to cytokinesis. Mitochondria regroup around the divided nuclear material. Endoplasmic reticulum cisternae remain expanded and outfolded stacks of peripheral membrane are complex.
Secondary spermatocyte Cytokinesis is incomplete and results in secondary spermatocytes connected by an intercellular bridge (Fig. 14). These intercellular bridges are distinguished from the cytoplasmic bridges of spermatogonia by the lack of dense materials on the cell membrane and absence of a spindle remnant. Processes of sustentacular cells do not penetrate between the dividing cells. Sequential sections indicate that pairs of secondary spermatocytes connected by only one intercellular bridge do occur. FIG. 5. Primary spermatocyte illustrating the large nucleus, nucleolus and chromoplast (arrow). Note homogeneous (neutral) lipid droplets, and scant endoplasmicreticulum. The heterogeneous lipid inclusion(asterisk) occurs in a sustentacularcell. x 24 300. FIG. 6. Part of a spermatid obliquely sectioned, showing mitochondria and the tubular sheath between them and the cell membrane, x 27 200.
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However, more numerous instances of spermatocytes connected by more than one intercellular bridge indicate that the second meiotic division rapidly follows the first. No interphase nuclei occur. Four spermatocytes are thus interconnected by intercellular bridges. A cytoplasmic lobe is pinched off from the group of spermatids close to the points of interconnections via intercellular bridges. This lobe lacks nuclear material but contains Golgi apparatus, endoplasmicreticulum, mitochondria, and vesicles (Fig. 14). It can be considered a residual body of superfluous cytoplasm. Dense bodies appear within the nuclear material first at the end of the first meiotic division (Fig. 12) and increase in numbers through the second division (Fig. 14). • The sequence of development to early spermatids is illustrated in Fig. 16.
Development of spermatids (spermateleosis) Spermatids do not remain connected by intercellular bridges of meiotic divisions, but are enclosed by processes of sustentacular cells until much of the differentiation of the nucleus and organelles is completed. The densities within nuclear areas of spermatocytes appear to be centers of chromatin condensation. They increase in number and size and ultimately coalesce (Fig. 17). The three nuclear regions identified in secondary spermatocytes persist throughout this condensation, so that three contiguous dense spherical masses ultimately form. A nuclear membrane is absenL The trilobed form of the nucleus can be identified by light microscopy of Feulgen or Giemsa-stained sperm (Fig. 18). A pair of centrioles lies close to the nucleus (Fig. 8). The multiple membrane stacks characteristic of the plasmalemma of spermatocytes do not appear after cell division has completed. However, other characteristic membranous organelles develop. As these organelles may be related to membranous bodies characteristic of other nematode sperm they will be referred to by the term introduced by Foor (16), i.e. membrane specializations. Early spermatids are
FIG. 7. Primary spermatocyte undergoing nuclear breakdown. Note expanded form of nuclear envelope and endoplasmic reticulum membranes. Arrows note points where nuclear envelope is not expanded. The cell is enclosed by fine processes of sustentacular cells. At one point the cell membrane of the spermatocyte is outfolded (of). g = Golgi apparatus, x 19 400. FIG. 8. One of a pair of centrioles composed of nine single tutules close to dense nuclear material in a late spermatid. × 63 000. FIG. 9. Primary spermatocyte immediately after breakdown of the nuclear envelope. Nuclear region (n) is of lower granular density, mitochondria are scattered, g = Golgi apparatus, of= outfoldings of cell membrane x 19 700. FIG. 10. Primary spermatocyte in which nuclear meterial (n) is ringed by mitochondria. Note expanded form of endoplasmic reticuluha. The spermatocyte is enclosed by cytoplasm of sustentacular cells that contains mitochondria (arrows) smaller than those of the spermatocyte, x 17 000. FIG. 11. Polar view of a dividing primary spermatocyte illustrating three areas of nuclear material (n) and spindle fibers converging to a point close to a centriole (arrow). Note expanded form of endoplasmic reticulum and outfoldings (of) of cell membrane, g = Golgi apparatus. × 22 300.
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characterized by containing many tubules (Figs. 15 and 17), 850-1 250 • in diameter. Tubules appear to collapse upon themselves forming concave vesicles with an intermembrane space of 260-380 A . These in turn appear to close upon themselves and fuse with adjacent units to give double membrane loops enclosing a portion of cytoplasm. These membrane specializations ultimately come to lie just below the cell membrane around the tail portion of the mature sperm (Figs. 20, 21, and 23).
Spermatozoa As nuclear material condenses to form the trilobed nucleus, the mitochondria remain encircling the nuclear region (Fig. 17). A sheath composed of tubules (each about 180-200 A in diameter) lies between the mitochondria and cell membrane (Figs. 6, 15, and 17). With the complete condensation of the nucleus, the spermatid begins to undergo its final change in form to that of the spermatozoon. This involves elongation of the cell and distribution of the membrane specializations peripherally in one half, arbitrarily referred to as the tail. The tubular sheath breaks down and mitochondria become distributed throughout the sperm. The mature sperm is about 14 × 2 #m and shows a separation by a slight constriction into a blunter head region and a more tapering tail (Fig. 19). The trilobed, Feulgen-positive nucleus lies in the head portion close to the middle of the sperm. Although mitochondria als0 occur, m u c h of the cytoplasm contains fi-glycogen granules. These are stained in electron microscope preparations by the modified PAS reaction and account for PAS-positive, diastase-sensitive staining of the sperm seen in light microscopy. Membrane specializations are closely packed around the periphery of the tail while mitochondria and some fl-glycogen occurs centrally. Within the lumen of the membrane specializations are fine filaments about 100 A thick (Figs. 22 and 23). In late spermatids and spermatozoa the cytoplasm contains an anastam0sing network of material that, especially near the cell periphery, appears tubular (Figs. 20 and 23). This does not clearly resemble agranular endoplasmic reticulum but may be only the fixation pattern of the glycogen-rich cytoplasmic matrix. Pseudopodia projec t irregularly from both head and tail of the sperm but more were found on the head. They were first seen in sperm in the lumen of the testis but occur in greater numbers on sperm in the vas deferens (Fig. 21). In phase-contrast light microscopy they appear as very fine threads. No motility was noted in sperm dissected into Tyrodes' physiological salt solution and examined by phase microscopy. Sperm seen in the uterus of a limited number of female worms resembled those in the vas deferens of males. Figure 24 illustrates stages in transformation of spermatids into spermatozoa. FIG. 12. Section through dividing primary spermatocyte. A sustentacular Cell encloses the spermatocyte, g=Golgi apparatus, n=nuclear material, sn =sustentacular cell nucleus, x 14 000. FIG. 13. A phagocytic vacuole in a sustentacular cell. x 5 600.
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Fro. 16. Diagrammatic summary of spermatid development. Spermatogonia (a) undergo mitotic divisions that form spindle bridges between daughter cells and interphase nuclei reform (b). Primary spermatocytes enlarge; the nuclear envelope and endoplasmic reticulum expands (c). After the nuclear envelope breaks down, chromatin is ringed by mitochondria (d). The first meiotic division is characterized by a cytoplasmic bridge connecting secondary spermatocytes (e). The second meiotic division follows giving a tetrad of spermatids connected by cytoplasmic bridges, and a residual body of cytoplasm is also pinched off (f). Outfoldings of the plasmalemma that develop first on the primary spermatocyte (c-d) and are present through to the tetrad stage (f), have been omitted from the diagram for clarity.
Sustentacular cells S u s t e n t a c u l a r cells envelop s p e r m a t o g e n i c cells up to the early s p e r m a t i d stage. T h e y are recognizeable b y their large p l e o m o r p h i c nuclei (especially w h e n fixed directly in o s m i u m ) a n d extensive c y t o p l a s m (Figs. 1 a n d 12). T h e nucleus has a large nucleolus a n d finely scattered c h r o m a t i n . Centrioles were seen close to the nucleus in a few s u s t e n t a c u l a r cells. E n d o p l a s m i c reticulum a n d G o l g i a p p a r a t u s occur as well as m i t o c h o n d r i a t h a t are smaller t h a n those in germinal cells (Fig. 10). L i p i d inclusions are b o t h in the f o r m of h o m o g e n e o u s d r o p l e t s ( p r o b a b l y n e u t r a l FIo. 14. Division stage of secondary spermatocytes showing spermatids (1-3) connected by cytoplasmic bridges (arrows) and a residual body (RB) containing three Golgi apparatuses (g), mitochondria, and light vacuoles. Small dense bodies occur in the nuclear region, x 13 600. FIG. 15. Spermatids containing large tubules. A dense nuclear body occurs in one spermatid and the sheath between mitochondria and cell membrane is indicated (arrows). Tubules appear to be collapsing to form membrane specializations, x 18 400.
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FIG. 17. Two spermatids showing condensation of nuclear material in one and collapsing tubules in the cytoplasm (arrows) × 31 000.
FI6. 18. Photomicrograph of a sperm stained with Giemsa showing the trilobed nucleus, x 1 360. FIO. 19. Phase-contrast photomicrograph of living sperm showing division into head and tail regions by slight constriction (arrow). x 1 170. 1 ~ . 20. Sperm at the periphery of the lumen of the testis. The tail region contains membrane specializations and mitochondria. Two of the three nuclear lobes are evident. Two basal laminas and a muscle cell form the wall of the testis, p s - p s e u d o c o e l o m , n=nucleus, x35 300.
16
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FIG. 24. Diagrammatic summary of spermatid differentiation into sperm. Nuclear material is initially dispersed with a few dense bodies within it. Chromatin condensation progresses to form a trilobed, dense, non membrane-bound nucleus in the sperm. Mitochondria are arranged around the nuclear region during this chromatin condensation and a fine tubular sheath lies external to them. The sheath disappears as mitochondria redistribute in the sperm. Irregularly expanded endoplasmic reticulum membranes of early stages may transform into tubular units that progressively collapse upon themselves and unite with neighbors to form membrane specializations in the tail of the sperm. Centrioles remain close to the nucleus. The sperm is separated by a slight constriction into head and tail regions. Pseudopodial projections are most common on the head region in sperm in the vas deferens. Beta glycogen (not shown) occurs in the cytoplasm of the head region and to a lesser extent in the taiI region of the mature sperm.
lipid) a n d m u l t i l a m i n a t e o r w h o r l e d s t r u c t u r e s ( p r o b a b l y p h o s p h o l i p i d ) . W i t h i n t h e m a s s of c o m p a c t cells w h e r e d i f f e r e n t i a t i o n of early s p e r m a t i d s is o c c u r r i n g , sustenta c u l a r cells h a v e fine cell p r o c e s s e s e n c l o s i n g t h e g e r m i n a l cells (Figs. 10, 11, a n d 12). I n s o m e areas, t h e v o l u m e of s u s t e n t a c u l a r cell c y t o p l a s m is larger. H e r e o c c u r e m p t y v a c u o l e s , a n d v a c u o l e s e n c l o s i n g v a r y i n g q u a n t i t i e s of d e n s e g r a n u l a r a n d m e m b r a n o u s m a t e r i a l s (Fig. 13). A l s o , e n c l o s e d b y s u s t e n t a c u l a r cells are large m a s s e s r a n g ing f r o m t h e size of r e s i d u a l b o d i e s of s p e r m a t o c y t e s to t h e size of e n t i r e g o n i a l cells. FIef. 21. Sperm in the vas deferens show numerous irregular pseudopodial projections. Arrow indicates a centriole, n - nuclei, x 25 600. FIG. 22. Section through some of the membrane specializations of the tail of sperm showing crosssectioned filaments (arrows) within them. x 74 200. FIG. 23. Section through two tails and a head region of sperm in the vas deferens showing longitudinal profile of a filament (arrow) within a membrane specialization. × 44 600.
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These have a dense matrix containing mitochondria and other remnants of organelles. They appear to be degenerated germ cells. Some of these occur also in the lumen of the testis among spermatozoa. Here they are not enclosed by sustentacular cells. DISCUSSION The sequence of sperm development in Capillaria hepatica agrees with the general gametogenic pattern of nematodes (44). The cell architecture of spermatogonia and spermatocytes in general agrees with those of similar stages in Parascaris equorum (12) and Rhabditis pellio (3). Spindle bridges formed during division of spermatogonia in C. hepatica resemble spindle bridges characteristic of mitotic divisions in somatic cells (13). In contrast, divisions of spermatocytes are characterized by the formation of cytoplasmic bridges. As spindle remnants are absent from these bridges it is possible that they allow more effective communication between daughter cells and may be important in synchronizing the development of interconnected germ cells (13). In mammals, up to 15 spermatids may be interconnected by intercellular bridges (32). From light microscopic studies of nematode spermatogenesis, interconnection of four spermatids giving rise to tetrad formations seems most common (29, 31, 44). In C. hepatica, four spermatids are briefly interconnected at the end of the second meiotic division. Roosen-Runge (38), Nieander (32), and Courot et al. (9) have suggested that synchrony of sperm development is controlled more through the action of sustentacular cells. In C. hepatica, sustentacular cells are closely applied to spermatogenic cells up to the early spermatid. Spermatogenesis in C. hepatica does not result in the formation of spermatophores. Within the testis, development of sperm does not appear to be highly synchronized but in general does resemble the sequence of differentiation seen in seminiferous tubules of vertebrates. The release of extraneous cytoplasm from nematode spermatocytes as a residual body has been noted previously (2, 3, 7, 8, 12, 17, 22, 29, 31, 40, 41). In C. hepatica, as in vertebrates, release of residual bodies and separation of spermatids occurs simultaneously (14). Spermiation (release of spermatids from sustentacular cells) occurs in mammals by swelling of cisternae of endoplasmic reticulum of the sustentacular cell (42, 43). In this process, spermatids are released while residual bodies remain trapped in the sustentacular cell. Sustentacular cells in the testis of C. hepatica contain materials resembling residual bodies as well as discrete phagocytic vacuoles. This suggests that material of the residual body is degraded and possibly reutilized in the testis. In areas with sustentacular cells and sometimes within the lumen of the testis among sperm, large dense cells occur. These seem to be degenerate germ cells of various stages of development. In the human fetal testis similarly degenerated
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germ cells occur (18), and it is suggested that their phagocytosis by Sertoli cells may contribute to the nutritional requirements of the testis. A similar phenomenon probably occurs in c. hepatica when degeneration initiates in cells associated with sustentacular cells. However, degeneration of later stages may sometimes result in release of these cells along with sperm. Throughout spermatocyte stages, the peripheral cell membrane is elaborately folded. Such folds may be important in uptake of nutrients from sustentacular cells. After separation of spermatids, the outfoldings are absent. Throughout spermatocyte stages in C. hepatica, mitochondria occur around the nuclear region. A similar nuclear-cytoplasmic association was noted for Parascaris equorum (12), Ascaris lumbricoides, Gnathostoma sp., and Dirofilaria immitis (17), and for Rhabditis pelIio (3). In C. hepatica the mitochondria remain closely associated with the nuclear region until condensation of nuclear material is complete, but they then disperse through the sperm. During the period of nuclear condensation the tubular sheath occurs around the mitochondria and may serve to retain the mitochondria in position. A similarly intimate relationship exists between mitochondria and condensing nuclear material in sperm of Hydra littoraIis (45). During the past decade the ultrastructure of sperm from several nematodes has been described (6, 12, 21, 25, 35, 48). Foor (17) characterized four sperm types-ascaroid, strongyloid, diotophymoid, and oxyuroid. As the sperm of C. hepatica does not conform to any of these, a fifth, trichuroid type may be appropriate. The only valid generalizations are that nematode sperm lack flagella, lack an acrosome, and form pseudopodia. The nucleus of sperm of C. hepatica resemble other nematode sperm in lacking a bounding membrane. The trilobed shape of the nucleus reflects the haploid chromosome number. A maximum of three chromatin regions were seen in each pole of dividing spermatocytes. Miller (29) found that chromosomes of Onchocerca volvulus and Wuchereria bancrofti remained distinct throughout spermatogenesis and in the spermatozoa. Centrioles in developing and mature sperm are atypical as they contain only nine single tubules rather than triplets of tubules. Similar centrioles have been found in Nippostrongylus brasiliensis and Ancylostoma duodenale (17, 21). Membrane specializations are characteristic organelles in nematode sperm. Those in C. hepatica do not resemble those of the other sperm types described by Foor (17). The several types of membrane specializations found might reflect differences in their mode formation. It was concluded that membrane specializations of Parascaris equorum, Panagrellus silusiae, and Rhabditis pellio developed from Golgi dictyosomes (3, 12, 35), although Foor (17) suggested that the similar-appearing membrane specializations of Ascaris lumbricoides might develop from mitochondria. The origin
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of the large-diameter tubules from which the membrane specializations differentiate in C. hepatica is not clear. As the irregularly expanded endoplasmic reticulum of earlier spermatocytes is not recognizeable in spermatids, it may have been transformed into the tubules. On the other hand, the membrane specializations of the sperm do resemble membrane loops in the peripheral cytoplasm of tick spermatocytes. These form from invaginations of the peripheral membrane that pinch off to give vesicles (4). Ultimately the membrane loops fuse to form the central vacuole and motile processes of the tick's sperm (37). The membrane specializations of C. hepatica resemble bodies in the sperm of the rotifer Asplanchna sp. (23). The possible role of membrane specializations as acrosomal elements (6, 12) has been eliminated by Foor's observations (16) that the sperm of Ascaris penetrates the oolemma by means of pseudopodia formed on the region of the sperm that lacks membrane specializations. Foor (17) has suggested that final maturation of sperm (capacitation) may occur in utero, and may involve opening of membrane specializations through the surface. Sperm seen around oocytes in females of C. hepatica resembled sperm in the male tract; membrane specializations had not opened to the surface. The identification of sustentacular cells in the testis of C. hepatica confirms Rauther's observations (36) of nongerminal tissue in the hologonic testis, and supports the parallel between these gonads and seminiferous tubules of vertebrates as noted by Chitwood and Chitwood (5) and Nigon (33). Sustentacular cells, known best in vertebrates as Sertoli cells, are referred to as nurse cells, nutritive cells, or support cells in many invertebrates (1, 24, 28, 30, 49). Sato et al. (39) identified supporting cells surrounding spermatogonia in the trematode Paragonimus miyazakii but Featherston (15) did not describe them from the cestode Taenia hydatigena. The role of sustentacular cells is probably 3-fold as in vertebrates--nutritional, regulatory and supportive. The first two of these roles have already been mentioned. The enclosing of spermatocytes by sustentacular cells and the elaborate outfoldings of the spermatocyte membrane may be related to nutrient transfer to the germ cells while phagocytosis of residual bodies and degenerating germ cells may indicate recycling of nutrients. The regulation or synchronization of gamete development may be mediated by either or both sustentacular cells or cytoplasmic bridge connections between spermatocytes. As the testis is a single tubular organ, germinal cells developing along its perimeter would be carried into the flow of sperm that pass through the central lumen unless they were retained in position. This is the supportive role of sustentacular cells. REFERENCES 1. ANDERSON,W. A., and ELLIS, R. A., Z. Zellforsch. Mikrosk. Anat. 77, 80 (1967). 2. ANYA,A. O., Parasitology, 56, 347 (1966). 3. BEAMS,H. W. and SEKHON,S. S., or. Ultrastruct. Res. 38, 511 (1972).
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44. WALTON,A. C., in CHITWOOD,B. G. and CHRISTIE,J. R. (eds.), An Introduction to Nematology, Section II. Leader Press, Babylon, NY, 1940.
45. WE1SSMAN,A., LENTZ,T. L. and BARRNETT,R. J., J. Morphol 128, 229 (1969). 46. WRIGHT,K. A., Can. J. ZooL 38, 167 (1961). 47. WRIGHT,K. A. and DICK, T. A., Z. Parasitenk. 40, 75 (1972). 48. WRIGHT, K. A., HOPE, W. D. and JONES, N. 0., Proc. Helminthol. Soc. Wash. 40, 30 (1973). 49. YASUZUMI,G., TANAKA,H. and TEZUKA,O., J. Cell BioL 7, 499 (1960).