Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

Veterinary Parasitology 187 (2012) 168–182

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Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis R.E.B. Hanna a,∗ , D. Moffett a , G.P. Brennan b , I. Fairweather b a b

Veterinary Sciences Division, Agri-Food and Biosciences Institute, Stormont, Belfast BT4 3SD, United Kingdom School of Biological Sciences, Queen’s University, Belfast BT7 1NN, United Kingdom

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Article history: Received 4 August 2011 Received in revised form 20 December 2011 Accepted 29 December 2011 Keywords: Fasciola hepatica Triclabendazole-sensitive and -resistant isolates Testis Light and electron microscopy Sustentacular tissue Heterophagy and apoptosis

a b s t r a c t In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Recent studies on the histology and cytology of the testis in flukes of triclabendazole-sensitive (TCBZ-S) Fasciola hepatica isolates have revealed that this tissue exhibits

∗ Corresponding author. Tel.: +44 0 2890 525615; fax: +44 0 2890 525767. E-mail address: [email protected] (R.E.B. Hanna). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.12.037

significant drug-induced changes as early as 24 h after administration of TCBZ to the host. These changes include depletion of germinal line cells from the testis tubules, vacuolation and development of characteristic rounded eosinophilic apoptotic bodies (Hanna et al., 2010a, in press; Toner et al., 2011). The nature of these changes is consistent with inhibition of mitosis and meiosis, possibly due to blockage of spindle formation by the postulated anti-tubulin activity of TCBZ (Lacey, 1988; reviewed by Fairweather and Boray, 1999). Following an aborted

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attempt to undergo nuclear segregation, it is postulated that a cascade of biochemical events is triggered in each dividing cell that ultimately leads to apoptosis (individual cell death) (Hanna et al., 2010). Apoptotic-type changes have also been described in the primary spermatocytes of untreated Cullompton isolate flukes, which, being triploid, are incapable of initiating normal meiotic division (Fletcher et al., 2004; Hanna et al., 2008). In order to verify that the morphological changes documented in histological sections of affected fluke testis coincide with endogenously-triggered individual cell death, commercially available kits were used to demonstrate and localise apoptosis by in situ labelling of endonuclease-induced DNA strand breaks (Hanna et al., 2010b). This approach drew attention to the occurrence in F. hepatica testis sections of endonuclease activity located in the tubules elsewhere apart from the apoptotic germinal line components. In vertebrate testis the occurrence and roles of Sertoli cells are well established (Junqueira et al., 1989). They associate intimately with cells of the germinal lineage in the lining of the seminiferous tubules, serving to support and nourish the developing spermatogonia, and to scavenge residual cytoplasm and effete or damaged spermatids and spermatozoa by a process of heterophagy, employing lysosomally derived hydrolytic enzymes including endonucleases. The presence of non-germinal line cells with functions analogous to the Sertoli cells in vertebrates has been recorded in Schistosoma mansoni by Otubanjo (1981). In a few other trematode species, these so-called sustentacular cells have also been recorded in the course of ultrastructural studies aimed primarily to document the processes of spermatogenesis and spermiogenesis (Robinson and Halton, 1982; Erwin and Halton, 1983). In F. hepatica, spermatogenesis, spermiogenesis and spermatozoon morphology were described in detail by Stitt and Fairweather (1990), but these authors did not include reference to non-germinal tissues. The present investigation was prompted by a need to clarify intercellular relationships and the dynamics of interaction between germinal and non-germinal line tissues within the testis tubules of F. hepatica, particularly with reference to intracellular and extracellular scavenging mechanisms triggered by nuclear damage. It is hoped that the study will increase our understanding of drugmediated effects on the reproductive system of trematodes, including F. hepatica, and aid in the characterisation of emerging drug resistance. 2. Materials and methods 2.1. Sources of material A range of flukes, derived from experimental infections in sheep, cattle and rats with metacercariae of TCBZ-S and TCBZ-R F. hepatica isolates was examined in this investigation. The history and TCBZ resistance characteristics of each experimental isolate, namely, Cullompton, Fairhurst, Oberon and Sligo, were summarised by Fairweather (2011). Flukes from rats and sheep were collected from the livers at least 10 weeks after infection by oral gavage with metacercariae obtained from laboratory-maintained colonies of Galba truncatula, while those from cattle were collected at

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least 16 weeks post-infection. Experimental sheep and cattle were euthanized by captive bolt stunning, followed by exsanguination and removal of the liver. Rats were euthanized using carbon dioxide prior to liver removal. Mature flukes were also examined from field cases of chronic fasciolosis. They were collected from the bile ducts of freshly dead carcases of sheep submitted to the Pathology Section of VSD, Stormont for post-mortem examination. 2.2. Tissue preparation for histology Flukes selected for examination were placed in a 10 cm2 plastic Petri dish and flat-fixed for 2–4 h underneath glass microscope slides using 10% (v/v) neutral buffered formalin. Thereafter, the flukes were free-fixed in 10% (v/v) neutral buffered formalin at 4 ◦ C overnight. Following this, each fluke was sliced into equal right and left halves along the median plane. The two halves were dehydrated through an ascending series of ethanol, cleared in Clearene (Surgipath Europe Ltd.) and embedded in wax blocks following conventional procedures, with the cut surfaces presented at the block face. Sections 3 ␮m in thickness were cut from each block face and stained with haematoxylin and eosin using standard histological protocols. Sections were examined and the testis profiles were photographed using a Leica DM LBZ microscope with a Nikon Coolpix 5000 camera system. 2.3. Tissue preparation for transmission electron microscopy Initially each fluke was flat-fixed under a microscope slide for 30 min at room temperature in 4% (w/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2). Transverse slices (3 mm in thickness) were then cut from the mid-body region, behind the visible mass of the uterus. Slices were free-fixed for 4 h in 4% (w/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) and subsequently transferred and stored in 5% sucrose buffer in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 ◦ C until further processing. Fluke slices were then post-fixed in 1% osmium tetroxide for 1 h prior to dehydration through an ascending series of ethanol to propylene oxide. The tissues were infiltrated and embedded in Durcupan epoxy resin (Sigma–Aldrich Co. Ltd., UK) which was polymerised for 48 h at 60 ◦ C. Using 1 ␮m thick sections examined by light microscopy to select appropriate areas of each block face, ultrathin sections (60–70 nm thick) were cut from the resin blocks using a Leica EM UC7 ultramicrotome, mounted on uncoated nickel grids, double-stained with uranyl acetate and lead citrate, and viewed in a Hitachi H-7000 electron microscope operating at an accelerating voltage of 100 kV. 3. Results 3.1. Light microscopy Spermatogenic line cells in the testis of flukes of the Cullompton isolate were represented only by primary, secondary and tertiary spermatogonia and primary spermatocytes (secondary spermatocytes, spermatids and

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mature spermatozoa being absent), and mature spermatozoa were lacking in 50% of the Sligo isolate flukes. In contrast, the histology of the germinal tissues in the testes of the remaining 50% of Sligo flukes and in the Oberon, Fairhurst and most of the wild-type flukes examined here was consistent with full virility, featuring normalappearing mature spermatozoa. A few wild-type flukes (approximately 3%) lacked normal mature spermatozoa in the testis tubules (Fig. 1a–c), in contrast to the majority, which contained numerous mature spermatozoa, often arranged in ‘rafts’ (Fig. 1d and e). Within each testis tubule of all the flukes examined, cells of the germinal line occupying the centre (‘lumen’) were surrounded by eosinophilic hyaline-to-granular material. This was most evident in profiles of tubules in which the cells were widely separated and mature spermatozoa were absent (Fig. 1a–c). In flukes with mature spermatozoa present, the latter often occupied and obscured the eosinophilic matrix (Fig. 1d and e). No nuclei were associated with the extracellular eosinophilic matrix. The cells in the centre of each tubule were identified as tertiary spermatogonia, primary (8-cell) and secondary (16-cell) spermatocytes and spermatids (32-cell) at various stages of development. Also present were numerous circular or oval profiles of residual cytoplasm, hereafter referred to as residual vacuoles. They remain after spermatozoa have been released from the spermatid mass, and were characterised by their predominantly pale-staining or vacuolated appearance, although they often contained granular material and a few twisted spermatozoon or spermatid nuclei. Residual vacuoles were particularly evident in the testis tubules of individuals with normal-appearing mature spermatozoa (Fig. 1d and e), and were less frequent and often poorly defined in those individuals where spermiogenesis was not completed successfully (Fig. 1a–c). The peripheral zone of each testis tubule was occupied by primary and secondary spermatogonia, often arranged in elongated but discontinuous clusters, between which the eosinophilic extracellular matrix material containing spermatozoa, spermatids and residual vacuoles often penetrated to the tubule wall (Fig. 1d and e). Elongated peripheral vacuoles, distinct from the residual vacuoles mentioned above and containing variable amounts of coarse or fine basophilic granular material, sometimes occurred between or below the clusters of spermatogonia, displacing the contents of the tubule from direct contact with the wall. These peripheral vacuoles were especially evident in flukes lacking fully developed spermatozoa

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(Fig. 1a–c). Comparison of sections examined at higher light microscope magnification with low power electron micrographs (Figs. 2a, b, 3a, b and 4a) revealed occasional large, rounded, pale-staining nuclei with prominent nucleoli located at the periphery of the tubules, often amongst the more densely-staining basophilic nuclei of the primary and secondary spermatogonia (Fig. 1a–e), but sometimes projecting into the peripheral vacuoles (Fig. 1a). While the boundaries of the spermatogonia were evident by virtue of the staining properties of these cells, it was not possible at the light microscope level to define the boundaries of the tissue containing the pale-staining nuclei. 3.2. Electron microscopy 3.2.1. Cell inter-relationships In all of the sections of fluke testis examined in the present study, regardless of type, the germinal cells at various stages of development (namely the primary, secondary and tertiary spermatogonia, the primary spermatocytes and, where present, the secondary spermatocytes, the spermatids and the mature spermatozoa) were found to be surrounded by a finely granular extracellular matrix of low, fairly homogeneous electron density. Within this matrix, transverse, sagittal and oblique sections of spermatozoa were frequently seen in those flukes where spermiogenesis was accomplished successfully (Fig. 2a). The matrix material with its content of germinal line cells, residual vacuoles and spermatozoa was present throughout the centre of each tubule, but often extended between the peripheral clusters of spermatogonia to the basal lamina of the tubule wall (Fig. 2a). Amongst the clusters of primary and secondary spermatogonia at the periphery of each testis tubule, occasional large euchromatic nuclei, each with a conspicuous nucleolus (if present in the plane of section), were seen (Figs. 2a, b, 3a, b and 4a). They were readily distinguishable from the relatively heterochromatic nuclei of the spermatogonia, and thus were more easily identified in electron micrographs than in light microscope preparations. Each nucleus was surrounded by a layer of cytoplasm of variable thickness that was characterised by its electron lucidity and by the presence of conspicuous dense organelles, primarily mitochondria and lysosomes. The cytoplasm of this tissue (hereafter referred to as sustentacular tissue) featured numerous extensions that ramified between the adjacent spermatogonia and abutted the extracellular matrix with its contained spermatozoa and

Fig. 1. (a–c) F. hepatica: histological sections of testis tubules of aspermic individuals from a field case. Primary and secondary spermatogonia (Sg1/2) occur in elongated clusters in the peripheral zone of the testis tubules with the spaces between often vacuolated (v). Nuclei of sustentacular tissue (unlabelled arrows) are occasionally seen in the spermatogonial clusters or associated with the peripheral vacuoles. The core or ‘luminal’ zone of each tubule contains tertiary spermatogonia (Sg3) together with primary and secondary spermatocytes (Sc2) and spermatids (Sp). Vacuolated residual cytoplasmic bodies (R), often associated with spermatids, are also apparent in this zone. The cells are supported in an amorphous hyaline eosinophilic extracellular matrix material (E). The testis tubules are enveloped by muscle cells, the nuclei of which are evident (Mu), and are supported by the surrounding parenchymal tissue (P). (d and e) F. hepatica: histological sections of testis tubules of normal spermatozoa-producing individuals from a field case. Sustentacular nuclei (unlabelled arrows) are occasionally seen amongst the peripheral clusters of primary and secondary spermatogonia (Sg1/2). Components such as primary spermatocytes (Sc1), secondary spermatocytes, spermatids (Sp) and vacuolated residual bodies (R) that represent the later stages in spermatogenesis and spermiogenesis occupy the core zone of the tubule and extend between the peripheral clusters of spermatogonia, to the periphery. Mature spermatozoa (Sz), often occurring in ‘rafts’, are suspended in the extracellular matrix material (E) and also penetrate towards the limiting wall of the tubule. Peripheral vacuoles (v) are less frequent than in aspermic individuals. The tubules are supported in parenchymal tissue (P).

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other components, where it protruded towards the peripheral zone of the tubule (Fig. 2a). The extracellular matrix was sometimes insinuated between the sustentacular cytoplasm and the tubule wall. Elsewhere the sustentacular cytoplasm lined the inner aspect of the testis tubule, its basal membrane closely applied to the underlying basal lamina of the testis wall (Fig. 2a and b), although the width of the intervening space was not uniform, and there was no formation of desmosome-like structures. Profiles of smooth endoplasmic reticulum were commonly seen in the sustentacular cytoplasm adjacent to the basal lamina, and sometimes the cisternae appeared to be continuous with the basal cell membrane (Fig. 2b). Sections of spermatozoon axonemes present in the extracellular matrix often appeared very close to the limiting membrane of the sustentacular tissue, and sometimes appeared within the sustentacular cytoplasm itself (Fig. 2b), apparently lacking an intact enveloping membrane. Where extensions of the sustentacular cytoplasm abutted spermatogonia, the limiting membranes of the two cell types were generally closely apposed, with only a narrow electron-lucid space between (Fig. 2a) (similar to the arrangement where two neighbouring spermatogonia abutted). However, the membranes separated in places, and the width of the intercellular space was not uniform. There was no evident densification of the apposed cell membranes, or formation of desmosomes, tight junctions or junctional complexes, such as were evident between apposed parenchymal cell membranes and muscle cell membranes in the tissue immediately beneath the tubule wall (Figs. 3b and 4a). Likewise, no evidence was seen of desmosomes, tight junctions or junctional complexes between adjacent sustentacular cytoplasmic extensions, perhaps indicating that the sustentacular cells constituted a peripheral syncytium, partially enveloping the spermatogonia and in places lining the inner aspect of the basal lamina. 3.2.2. Cytoplasmic features of the sustentacular tissue Within the cytoplasm of the sustentacular tissue, large, rather rounded and moderately electron-dense mitochondria each with a few conspicuous cristae, were abundant, often occurring in clusters in the cytoplasmic extensions between spermatogonia or close to the nucleus (Fig. 2a and b). Also present, usually close to the nucleus, were ribosomes and cisternae of granular

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endoplasmic reticulum, the latter sometimes distended into sac-like structures by homogeneous and moderately electron-dense contents (Fig. 3a). In these areas, poorly organised Golgi fields were also evident featuring irregularly sized spherical, oval or elongated membrane-bound presumed primary lysosomes with rather heterogeneous electron-dense contents (Fig. 3a and b). In the sustentacular cytoplasm, secondary and tertiary lysosomes were also abundant, but generally they were not common in areas where cisternae of granular endoplasmic reticulum and Golgi fields were prevalent (as in Fig. 3a and b). Secondary and tertiary lysosomes were much larger than primary lysosomes, and tended to be irregular in shape with heterogeneous contents featuring areas of moderate electron density interspersed with dense granular or fibrous material (Fig. 4a–c). Secondary lysosomes were considered to be those in which the content was mainly of moderate density, while tertiary lysomes had predominantly dense content, often featuring numerous randomly orientated dense rod-like structures (Fig. 4b and c). Tertiary lysosomes with uniformly electron-dense contents or dense membranous whorls were considered to be cytosegrosomes. Mitochondria were generally abundant in areas of cytoplasm where lysosomal activity was evident (Fig. 4b and c), and sometimes sections of spermatozoon axonemal complexes were seen naked in the sustentacular cytoplasm alongside secondary and tertiary lysosomes (Fig. 4c). 3.2.3. Cytoplasmic features of aspermic flukes In the testis tubules of approximately 50% of flukes of the Sligo isolate, where spermiogenesis did not proceed to completion and normal mature spermatozoa were not seen, the sustentacular cytoplasm was often found to envelop apparently apoptotic cells. The latter had conspicuously dense cytoplasm, while the nuclear material appeared fragmented and highly condensed (Fig. 5a). The apoptotic cells were rounded, and the limiting membrane was sometimes breached, with vesiculation of the contents. Mitochondria and lysosomal components of the sustentacular cytoplasm surrounded the apoptotic cell bodies, some of which were partially isolated within an electronlucid vacuole (Fig. 5a). Within the sustentacular cytoplasm, portions of cells containing condensed nuclear material appeared, and fragments of axonemal complexes, apparently lacking an intact investing membrane, were closely

Fig. 2. (a) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. The periphery of the testis tubule is marked by the basal lamina (BL), beneath which the blocks of myofibrils (Mu) of the muscle cells are located. Near the periphery of the testis tubule a nucleus (Tn) of the sustentacular tissue lies in a thin layer of electron lucid cytoplasm (Tc), extensions of which surround and protrude between spermatogonia (Sg) that juxtapose the sustentacular tissue. In contrast to the euchromatic sustentacular nucleus with its prominent nucleolus, the nuclei of the spermatogonia are irregular and heterochromatic. Profiles of spermatozoon axonemes (Sz) are evident within the moderately electron dense extracellular matrix material (E) which abuts the sustentacular tissue, and in places extends between the sustentacular tissue and the basal lamina. Within the sustentacular cytoplasm mitochondria (m) are abundant close to the nucleus, and also in the extensions of sustentacular cytoplasm between the spermatogonia. Inset shows apposing membranes of the sustentacular cytoplasm (single arrows) and spermatogonia. Note that the space between the membranes is not completely uniform, and lacks densification. For comparison, the apposing membranes of the two spermatogonia are labelled X. (b) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. The limiting membrane of the sustentacular cytoplasm (Tc) abuts the basal lamina (BL) of the testis wall, but the membranes do not form desmosomes, and the space between is not completely uniform. Mitochondria (m) are common close to the sustentacular nucleus (Tn). Profiles of spermatozoa and their axonemes (Sz) occur in the extracellular matrix material (E) and apparently also within the sustentacular cytoplasm, close to the nucleus. The boundary between the sustentacular cytoplasm and the interstitial matrix (unlabelled arrow) is often indistinct. Inset shows cisternae of smooth endoplasmic reticulum (ser) which are common in the sustentacular cytoplasm in this area, some appearing to be continuous with the limiting membrane (unlabelled arrow). Mu = muscle; Sg = spermatogonium.

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associated with secondary and tertiary lysosomes, which were particularly abundant in these sections (Fig. 5b). The content of the secondary and tertiary lysosomes featured numerous randomly orientated rod-like structures (Fig. 5b), as described above (Section 3.2.2). 3.2.4. Residual vacuoles The large, apparently vacuolated bodies of residual cytoplasm that featured prominently in light microscope sections of normal sperm-producing flukes (Fig. 1d and e) were also identified at the electron microscope level. Each of these vacuoles was delimited by an envelope comprising flattened cisternae of granular endoplasmic reticulum of various widths and with homogeneous moderately electron-dense contents. These cisternae were arranged end-to-end with pore-like discontinuities between adjacent cisternae, often bridged by a single membrane (Figs. 5c and 6). The bridging membrane was considered to represent the original limiting plasma membrane of the spermatocyte, to which the cisternae of granular endoplasmic reticulum were closely apposed. Occasionally, late spermatids, not yet released, were seen attached to a sector of the peripheral bounding envelope of a residual vacuole (Fig. 6). Within each residual vacuole, the cytoplasm was characteristically electron-lucid and often the few organelles present were widely scattered in an apparently vacuolated matrix. Mitochondria were swollen and rounded with inconspicuous cristae. Irregular cisternae of smooth and granular endoplasmic reticulum often appeared swollen with homogeneous and moderately electron-dense contents, resembling those seen in the sustentacular cytoplasm. Primary, secondary and tertiary lysosome-like bodies were apparent, and membranous whorls, probably representing the end-products of lysosome-mediated breakdown of organelles, were also seen. 4. Discussion 4.1. Cell inter-relationships The most detailed account of the ultrastructure of spermatogenesis and spermiogenesis in F. hepatica was provided by Stitt and Fairweather (1990). In the present study, cells of the germinal line in all stages of development (primary, secondary and tertiary spermatogonia, primary and secondary spermatocytes, spermatids and mature spermatozoa) were recognised in Oberon, Fairhurst and most wild-type flukes, but stages subsequent to primary

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spermatocytes were absent from the triploid Cullompton isolate flukes (Fletcher et al., 2004; Hanna et al., 2008), while 50% of the Sligo flukes and a small number of wildtype flukes lacked mature spermatozoa. The histological features of the testes of wild-type flukes and flukes of various TCBZ-S and TCBZ-R isolates, including Cullompton, Sligo and Oberon, were described by Hanna et al. (2008, 2010a, in press). Previous electron microscope studies on the testis of F. hepatica have concentrated on cells of the germinal line, and little attention has been given to cells of the somatic or non-germinal line. However, several authors have reported non-germinal tissues within the testes of other trematodes. The species examined include S. mansoni (Otubanjo, 1981), Corrigia vitta (Robinson and Halton, 1982) and Bucephaloides gracilescens (Erwin and Halton, 1983). By analogy with similar non-germinal Sertoli cells in the testes of vertebrates (for review see Junqueira et al., 1989), these cells have been designated sustentacular cells. Based on their morphology and close association with spermatogonia and other germinal line cells, nutritive, supportive and phagocytic functions have been ascribed to them. In this study, sustentacular tissue was identified in the testis tubules of F. hepatica. No evidence was found of junctional complexes, tight junctions or desmosomelike structures between apposing cytoplasmic extensions in the sustentacular tissue, between these extensions and cells of the germinal line, or between the basal limiting membrane of the sustentacular tissue and the basal lamina of the tubule wall. Unlike the situation in the testis of vertebrates (Junqueira et al., 1989), it is possible that the sustentacular tissue in F. hepatica testis is a syncytium, as in B. gracilescens (Erwin and Halton, 1983). Conceivably, the lack of junctional complexes between the sustentacular tissue and the germinal line cells is to permit relative mobility and migration of the germinal line components from their origin at the periphery of the tubule, through the central region as development progresses, to ultimate evacuation via the vasa efferentia. Elsewhere in the tissues of F. hepatica where, for example, parenchymal extensions make contact with gut, excretory system and muscle cells, specialised dense regions of the apposed membranes form desmosome-like structures, presumably to impart structural stability while permitting exchange of ions, metabolites and excretory products (Threadgold and Gallagher, 1966; Gallagher and Threadgold, 1967; reviewed by Fairweather et al., 1999). The disposition of the sustentacular tissue in the testis has similarities with the arrangement of ‘nurse’ cells in the vitelline follicles of F. hepatica, which have a role in support, nutrition and

Fig. 3. (a) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. Within the sustentacular cytoplasm (Tc) are distended cisternae of granular endoplasmic reticulum (ger) containing homogenous, moderately electron dense material. These are often associated with Golgi fields (G) lying close to the nucleus (Tn). Primary lysosomes (L1) are abundant around the Golgi fields and elsewhere throughout the cytoplasm. The base of the sustentacular tissue is apposed to the infolded basal lamina (BL) of the testis wall, beneath which myofibrillar blocks of muscle cells (Mu) occur. Inset shows detail of a Golgi field (G) close to the sustentacular nucleus (Tn), with associated distended and flattened cisternae of granular endoplasmic reticulum (ger), ribosomes (r) and lysosomes (L1). (b) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. Sustentacular cytoplasm (Tc) containing primary lysosomes (L1) Golgi fields (G) and mitochondria (m) abuts the basal lamina (BL) at the periphery of a testis tubule. Extensions of the sustentacular cytoplasm penetrate between juxtaposing spermatogonia (Sg). A muscle cell with a prominent nucleus (Mu) lies beneath the basal lamina, and densified desmosome-like junctional complexes (arrowed) occur between the muscle cell and extensions of the parenchyma (P). Inset shows detail of a Golgi field (G) with associated ribosomes (r) and primary lysosomes (L1) close to the sustentacular nucleus (Tn).

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metabolic exchange for the developing and differentiating vitelline cells (Irwin and Threadgold, 1970; reviewed by Fairweather et al., 1999). By analogy with the arrangement of germinal and non-germinal tissue in vertebrate seminiferous tubules (Junqueira et al., 1989), the testis tubule of F. hepatica may be considered to comprise two relatively distinct, yet interpenetrating compartments, illustrated diagrammatically in Fig. 7: a. A peripheral zone, comprising the testis wall muscle cells, the basal lamina to which they are anchored and the inner sustentacular tissue which is supported by (but apparently not anchored to) the basal lamina. The sustentacular tissue partially envelops supports and nourishes the primary and secondary spermatogonia during the initial divisions of mitosis, and allows the resulting tertiary spermatogonia to migrate inwards towards the centre of the tubule, propelled by the pressure of the dividing cells beneath. b. A central zone containing tertiary spermatogonia and subsequent developmental stages, namely, the primary and secondary spermatocytes, spermatids, mature spermatozoa and residual vacuoles. These cellular components are supported in an extracellular matrix, which most probably comprises a protein-rich seminiferous fluid. The components of this central zone penetrate towards the periphery of the tubule, bulging between the clusters of primary and secondary spermatogonia. The fluid extracellular matrix provides a medium to support the maturing spermatozoa on their release from the cytosome, the latter remaining as a residual vacuole. The extracellular matrix probably facilitates active movement and propulsion of the spermatozoa towards the vasa efferentia and vasa deferentia in response to waves of contraction generated by the muscle blocks in the wall of the tubule. 4.2. Cytoplasmic features of the sustentacular tissue 4.2.1. Mitochondria and micromolecular flux In the cytoplasm of the sustentacular tissue, the appearance of numerous large mitochondria is consistent with a high level of metabolism, and with the support of energy-demanding processes such as transport of ions and micromolecules into and away from the testis tubules. Bearing in mind the extensive development of the testis system and the considerable output of spermatozoa by

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F. hepatica, there is clearly a high influx of micromolecular nutrients, and efflux of the waste products of metabolism. These activities are probably sited at the basal plasma membrane of the sustentacular tissue, and at the juxtaposed membranes of the sustentacular tissue and the germinal line cells, consistent with the observed distribution of mitochondria in the cytoplasmic extensions of the former. It is likely that the sustentacular cytoplasm acts as a conduit for micromolecular nutrients and metabolites by virtue of its resident metabolic capability. 4.2.2. GER-Golgi and protein/glycoprotein synthesis Mitochondria were also concentrated near the sustentacular nuclei and the presence of granular endoplasmic reticulum (GER)-Golgi fields in the same location provides evidence of protein or glycoprotein synthesis. Bearing in mind the co-occurrence of numerous secondary and tertiary lysosomes in the sustentacular cytoplasm, it is likely that this synthetic activity represents generation of hydrolytic enzymes and their compartmentation in primary lysosomes for subsequent use in intracellular digestion. Halton (1967), using histological techniques, recorded a moderately high level of acid phosphatase enzyme activity in the testes of F. hepatica, which is consistent with the occurrence of intracellular digestion. However, by analogy with the Sertoli cells of vertebrates (Junqueira et al., 1989), it is also possible that protein-containing macromolecules are secreted by the sustentacular tissue of F. hepatica into the extracellular matrix in the centre of the tubule to facilitate spermatozoon transport, and some such molecules may have hormonal or pheromonal roles. Hanna et al. (2011) postulated a pheromonal involvement in the final stages of maturation and onset of reproductive function of flukes in the bile duct of rats. Furthermore, steroid hormone-secreting cells (including the Sertoli cells) that are found in the testis, ovary and adrenal cortex of vertebrates characteristically have marked development of the smooth endoplasmic reticulum and numerous large mitochondria (Junqueira et al., 1989). These features were also noted in the sustentacular tissue of F. hepatica testis. 4.2.3. Lysosomes and heterophagy Secondary and tertiary lysosome-like organelles are a prominent feature of the cytoplasm of the sustentacular tissue in F. hepatica, and it is likely that they represent stages in heterophagic digestion of effete spermatozoa, spermatids and other residual products of spermatogenesis

Fig. 4. (a) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. Within the sustentacular cytoplasm (Tc) mitochondria (m), secondary and tertiary lysosomes (L3) and occasional primary lysosomes (L1) are present. The sustentacular cytoplasm abuts the basal lamina (BL) of the testis wall, beneath which extensions of parenchyma form desmosome-like tight junctions (arrowed) with muscle cell processes containing myofibrillar blocks (Mu). Spermatogonia (Sg) surround the sustentacular tissue, which features a large euchromatic nucleus (Tn). (b) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. In the sustentacular cytoplasm (Tc) close to a nucleus (Tn), mitochondria (m) are abundant, as are secondary (L2) and tertiary (L3) lysosomes. Abutting the sustentacular cytoplasm is an area of extracellular matrix material (E) containing profiles of spermatozoa (Sz). The boundary between the sustentacular cytoplasm and the matrix material is not distinct in this area. (c) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. Mitochondria (m), secondary (L2) and tertiary (L3) lysosomes are present in the sustentacular cytoplasm (Tc), as apparently is the axoneme of a spermatozoon (Sz). A primary lysosome (L1) is also evident. Note the fibrillar nature of the contents of the secondary and tertiary lysosomes which has morphological similarities to the lamellar sheets of chromatin seen in advanced spermatid nuclei. Inset shows close association between a spermatozoon axoneme (arrowed) and a secondary/tertiary lysosome (L2) in the sustentacular cytoplasm (Tc).

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Fig. 6. F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Oberon isolate. A cytoplasmic residual body (R) surrounded by extracellular matrix material (E) containing numerous profiles of spermatozoa (Sz). Several fully differentiated spermatids, about to detach from the residual body, are seen at their sites of origin (unlabelled arrows). The residual body is partially bounded by flattened cisternae of granular endoplasmic reticulum (ger). The pore-like discontinuities between adjacent cisternae are often bridged by a single membrane. Similar cisternae are also visible within the cytoplasm, associated with swollen mitochondria (m) and lysosomes (L) representing various stages in intracellular digestion. Inset shows detail of the bounding cisternae of granular endoplasmic reticulum, with ribosomes attached to both the inner and outer aspects (arrows).

and spermiogenesis that occur actively in the central zone of the testis tubule. This view is supported by the finding of axoneme fragments within the cytoplasm of the sustentacular tissue, closely associated with presumed heterophagosomes. The resemblance of the rod-like contents of the secondary and tertiary lysosome-like bodies in the sustentacular cytoplasm to the lamellar sheets of condensed chromatin in late spermatid nuclei described

by Stitt and Fairweather (1990), provides further evidence of the scavenging role of the sustentacular tissue, but the mechanism of uptake of effete spermatozoa and spermatids from the extracellular matrix is unclear. It is likely that residual material, damaged spermatozoa and spermatids undergo phagocytosis at the bounding membrane of the sustentacular tissue, and are initially brought into the cytoplasm within membrane-bound heterophagosomes.

Fig. 5. (a) F. hepatica: transmission electron micrograph of testis tubule of aspermic Sligo isolate. Two apparently apoptotic cells of the spermatogenic lineage (Sa) lie close to the basal lamina (BL) of the testis wall and are partially enveloped by sustentacular cytoplasm (Tc) bearing numerous rather rounded and vesiculated mitochondria (m). The apoptotic cells have fragmented nuclei (n) and electron dense cytoplasm which appears vesiculated in some areas (unlabelled arrow), perhaps where the integrity of the limiting membrane is disrupted. The sustentacular cytoplasm is vacuolated (v) in some areas abutting the apoptotic cells, and contains numerous irregular secondary and tertiary lysosomes (L3) with cytoplasmic and nuclear fragments (F), probably originating from cells of germinal lineage. Extracellular matrix material (E) partially surrounds the sustentacular tissue and apoptotic cells. Mu = muscle; P = parenchyma. (b) F. hepatica: transmission electron micrograph of testis tubule of aspermic Sligo isolate. A fragment of cytoplasmic and nuclear material (F) probably originating from a germinal line cell, lies close to a secondary lysosome (L2) in the sustentacular cytoplasm. The secondary lysosome (L2) contains numerous randomly orientated rod-like structures. There is no clearly defined membranous boundary between extracellular matrix material (E) containing spermatozoon axonemes (Sz) and the sustentacular cytoplasm. (c) F. hepatica: transmission electron micrograph of testis tubule of spermatozoa-producing Fairhurst isolate. A cytoplasmic residual body (R), remaining after the spermatids have been released as spermatozoa (Sz) into the surrounding matrix material (E). The residual body is partially bounded by cisternae of granular endoplasmic reticulum (ger), variously distended by homogenous moderately electron dense material. The pore-like discontinuities between adjacent cisternae are often bridged by a single membrane (unlabelled arrows). Similar granular endoplasmic reticulum cisternae also occur within the cytoplasm of the body, and are associated with lysosomes (L) and swollen mitochondria (m).

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Fig. 7. F. hepatica: simplified diagram to illustrate the arrangement of germinal line cells and sustentacular tissue at the periphery of the testis tubule. The cytoplasm of the sustentacular tissue (Tc, shaded) extends between spermatogonia (Sg) in the vicinity, providing support and probably acting as a conduit for the inward flux of nutrients across the basal lamina (BL) and muscle tissue (Mu) from the parenchyma (P), as well as for the outward movement of excretory metabolites. Close to the sustentacular nucleus (Tn) are cisternae of granular endoplasmic reticulum (ger) and Golgi fields (G), which produce proteinaceous secretory bodies including primary lysosomes (L1). The latter fuse with phagocytosed material such as damaged spermatozoa and residual debris (unlabelled arrows) from the extracellular matrix material (E). Heterophagic digestion within the sustentacular cytoplasm results in the formation of secondary and tertiary lysosomes (L3) and cytosegrosomes. Mitochondria (m) are abundant in the sustentacular cytoplasm. Towards the core of the testis tubule, supported by the extracellular matrix, later stages in spermatogenesis such as primary (Sc1) and secondary (Sc2) spermatocytes occur. Spermatids (Sp) ultimately detach from the residual cytoplasmic bodies (R), and the maturation of spermatozoa (Sz) may involve an association with sustentacular tissue.

Breakdown begins when primary lysosomes containing hydrolytic enzymes are fused with the heterophagosomes. Within the resulting secondary lysosomes, the phagocytosed components are progressively degraded. Potentially useful micromolecules are transported back into the sustentacular cytoplasm, while insoluble waste products accumulate within tertiary lysosomes and cytosegrosomes. Key aspects of this process are illustrated diagrammatically in Fig. 7. While F. hepatica has access to an abundant supply of food in the form of mammalian blood, and therefore may not be expected to rely on scavenging of micromolecular nutrients through a heterophagic system, it nevertheless has no ability to synthesise purine nucleotides de novo (Berens et al., 1995; Tielens, 1999). Considering the high demand for DNA synthesis in spermatogenesis, and the

limited supply of nucleated cells in mammalian blood, scavenging of nucleotides by heterophagy of effete spermatozoa and spermatids in the sustentacular tissue may be necessary to augment the output of spermatozoa in F. hepatica. In vertebrate testes, the Sertoli cells are believed to have a role in maturation of the spermatozoa. Late spermatids, although linked by cytoplasmic bridges with others derived from the same spermatogonium, develop relatively independently and do not break away from large cytoplasmic residual vacuoles, as is the case with F. hepatica and many other invertebrates. They associate with the apical ends of Sertoli cells which ingest fragments of residual cytoplasm from the spermatids by heterophagy and break them down intracellularly by a lysosome-mediated process (Tindall et al., 1985; Junqueira et al., 1989). In F. hepatica, a similar

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process may operate, in which maturation of individual spermatozoa is facilitated by a heterophagic ‘clean-up’ process mediated by the sustentacular tissue, but the large residual vacuoles which remain do not appear to be engulfed in their entirety by the sustentacular tissue. A further role for heterophagy in the testis tubules of F. hepatica may lie in the generation of osmotic gradients between the parenchyma and the testis tubule. An increase in the concentration of micromolecules within the extracellular matrix of the tubule, originating from active heterophagic digestion in the sustentacular cytoplasm, might result in the movement of water into the tubule from the surrounding tissues along an osmotic gradient. This might contribute to the fluidity of the extracellular matrix, facilitating spermatozoon mobility and transport. 4.3. Cytoplasmic features of aspermic flukes Approximately 50% of flukes of the Sligo TCBZ-R isolate have been shown to have defective spermiogenesis and, in these flukes, mature spermatozoa are not seen in the testis tubules (Hanna et al., 2008). Meiotic dysfunctionality and aspermy in triploid and in diploid flukes has also been described amongst Japanese and Korean fluke populations (Agatsuma et al., 1994; Terasaki et al., 2000, 2001). Amongst the populations of wild-type flukes examined in this study, a small percentage also exhibited abnormal spermiogenesis, and in them, like the Sligo flukes, no normal spermatozoa were present in the testis tubules. The sustentacular cytoplasm in the Sligo flukes was found to envelop numerous apparently apoptotic cells, the identity of which was uncertain, but which may have been spermatids or spermatocytes, on the basis of the presence within them of multiple nuclear fragments. The degraded products of apoptosis of these cells apparently underwent heterophagy in the sustentacular cytoplasm, with resultant increase in the numbers of secondary and tertiary lysosomes. The increase in heterophagic activity in the sustentacular cytoplasm of the aspermic flukes probably reflects augmentation of the intracellular scavenging process in response to the large numbers of defective spermatids present in these individuals. The peripheral vacuolation noted in the light microscope preparations may have resulted from water flooding into the sustentacular cytoplasm following an osmotic gradient engendered by the increased heterophagic activity. 4.4. Residual vacuoles The occurrence of elements suggestive of a GER-Golgilinked lysosomal digestive process within the residual vacuoles after the completion of spermiogenesis probably reflects the operation of an intracellular scavenging process to recycle nutritive molecules and eliminate dead space within the testis tubules. However, the vacuolation of these structures evident in light microscope preparations suggests that here, as in the sustentacular cytoplasm, water may enter from the surrounding tissues along an osmotic gradient. Once again, there may be a role in storing or channelling water into the extracellular matrix to increase fluidity and facilitate spermatozoon motility and transport.

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The redundant cytoplasmic residual vacuoles, which are numerous and conspicuous components of testis sections viewed by light microscopy, may therefore have a final role in osmoregulation within the testis tubules. 4.5. Conclusion Sustentacular tissue, probably organised as a syncytium, has been identified in the peripheral zone of the testis tubules in F. hepatica. The ultrastructural features of this tissue indicate likely roles in support, nutrition, and metabolic exchange for the germinal line cells. Protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, and some molecules may act as pheromones. The sustentacular tissue scavenges effete spermatozoa and spermatids by heterophagy, enabling re-cycling of nutrient molecules, eliminating dead-space in the testis tubules, and possibly facilitating influx of water to the seminiferous fluid by enhancing the osmotic gradient. Heterophagy may also aid spermatozoon maturation. In aspermic flukes, heterophagic activity in the sustentacular tissue is apparently enhanced to degrade the large numbers of dysfunctional spermatids and apoptotic germinal line cells. Residual vacuoles carry out endogenous lysosome-mediated intracellular digestive processes and, like the sustentacular tissue, may contribute to influx of water by osmosis. Acknowledgements We gratefully acknowledge the co-operation and expertise of the staff of the Histopathology Section, the Pathology Section and the Photographic Unit, AFBI. Certain elements of this work were carried out under the auspices of the EU Sixth Framework DELIVER programme, Grant No. food-CT200X-023025. References Agatsuma, T., Terasaki, L., Yang, L., Blair, D., 1994. Genetic variation in the triploids of Japanese Fasciola species, and relationships with other species in the genus. J. Helminthol. 68, 181–186. Berens, R.L., Krug, E.C., Marr, J.J., 1995. Purine and pyrimidine metabolism. In: Marr, J.J., Muller, M. (Eds.), Biochemistry and Molecular Biology of Parasites. Academic Press, London, UK, pp. 89–117. Erwin, B.E., Halton, D.W., 1983. Fine structural observations on spermatogenesis in a progenetic trematode, Bucephaloides gracilescens. J. Parasitol. 13, 413–426. Fairweather, I., 2011. Liver fluke isolates: a question of provenance. Vet. Parasitol. 176, 1–8. Fairweather, I., Boray, J.C., 1999. Mechanisms of fasciolicide action and drug resistance in Fasciola hepatica. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Wallingford, Oxon, UK, pp. 225–276 (Chapter 7). Fairweather, I., Threadgold, L.T., Hanna, R.E.B., 1999. Development of Fasciola hepatica in the mammalian host. In: Dalton, J.P. (Ed.), Fasciolosis. CABI Publishing, Wallingford, Oxon, UK, pp. 47–111 (Chapter 3). Fletcher, H.L., Hoey, E.M., Orr, N., Trudgett, A., Fairweather, I., Robinson, M.W., 2004. The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica. Parasitology 128, 69–72. Gallagher, S.S.E., Threadgold, L.T., 1967. Electron microscope studies of Fasciola hepatica. II. The interrelationship of the parenchyma with other organ systems. Parasitology 57, 627–632. Halton, D.W., 1967. Studies on phosphatase activity in Trematoda. J. Parasitol. 53, 46–54.

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