Ultrastructural study of vitellogenesis and oogenesis of Crepidostomum metoecus (Digenea, Allocreadiidae), intestinal parasite of Salmo trutta (Pisces, Teleostei)

We describe the vitellogenesis and oogenesis of Crepidostomum metoecus from Salmo trutta collected in Corsica. This is the first study conducted in the Allocreadiidae family. The maturation of C. metoecus vitellocytes comprises four different stages depending on organelle content. The follicular vitellarium is surrounded by a basal lamina. Vitellocytes are randomly distributed into the vitellarium, although fully mature vitellocytes are found in the center of the follicle. During maturation, the nucleo-cytoplasmic ratio decreases, whereas synthetic activity increases. Fully mature vitellocytes are filled with β-glycogen particles and shell globule clusters. Compared to other trematodes studied, C. metoecus possesses a large amount of nutritive reserves for the developing embryo and high quantities of material for the developing shell. Oocyte maturation takes place in four stages: oogonia, primary oocytes, developing oocytes, and mature oocytes. Developing oocytes enter the zygotene-pachytene stage of the first meiotic division recognizable by the presence of synaptonemal complexes in the nucleoplasm. The low protein composition of mature oocytes associated with the large nutrient content of vitellocytes of C. metoecus enables us to consider that oocytes do not take part of the nutrition of the future embryo of the miracidium. A cytochemical test (Thiéry method) allowed us to detect the presence of polysaccharides and glycogen during maturation of these two cell types.


Introduction
The absence of fossils is a feature in Platyhelminthes, especially parasites. Therefore, to understand the relationships between species and their phylogeny, it is possible to research extant taxa, and include morphological and ultrastructural data, and more recently molecular analysis findings. Although the number of ultrastructural studies of vitellogenesis in digeneans has increased, several species (or families) have not been significantly examined to date. In fact, among the 18,000 digenean species [3,10] that have been described, fewer than 20 were studied for their vitellogenesis [7, 8, 12-15, 21-24, 30, 32, 37, 42, 45, 46]. Crepidostomum metoecus (Braun, 1900) is already listed in several studies as a parasite of brown trout in European countries [39]. According to Quilichini et al. (2007), the wide geographical distribution has led to diversity of intermediate hosts [39]. The vitelline cells provide the material necessary for the formation of the eggshell and the essential nutrient material for the development of the future embryo. The oogenesis of trematodes has been the subject of several studies by light and electron microscope [2,4,6,16,18,20,25,29,31,33,40,43,44,48,49,[50][51][52][53]. The female reproductive system of Platyhelminthes shows great morphological variability with significant differences in anatomical organization and cell structure between taxa. Platyhelminthes have been subdivided into two levels of organization, according to the female reproductive system. The Archoophora possess homocellular female gonads consisting of only germaria with oocytes which produce both yolk and eggshell forming precursors. The Neoophora are characterized by heterocellular female gonads composed of an ovary and vitelline glands. The neoophoran digenean Crepidostomum metoecus belongs to the Allocreadiidae family (Looss, 1902). The Allocreadiidae are relatively small digeneans that are parasites of the digestive system of teleosts, and occasionally snakes, salamanders, and frogs. The present study shows, for the first time, the ultrastructure of the female gonads of an Allocreadiidae. The aim of this study was to describe the ultrastructural characteristics of oocytes and vitellocytes during their differentiation.

Materials and methods
Adult specimens of Crepidostomum metoecus were collected live from the intestine of naturally infected Salmo trutta (Linnaeus, 1758) collected in Corsica. Worms were removed from their hosts, fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, postfixed in cold (4°C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide, embedded in Spurr, and polymerized at 60°C for 24 h. Ultrathin sections (60-90 nm) of the worms, at the level of the ovary or vitelline follicles, were cut on an ultramicrotome (PowerTome PC, RMC Boeckeler). Sections were placed on 300-and 200-mesh copper and gold grids. Sections on copper grids were stained with uranyl acetate and lead citrate [41]. Sections on gold grids were stained with periodic acid, thiocarbohydrazide, and silver proteinate [47]. This technique was used to detect glycogen. Sections were examined on a Hitachi H-7650 transmission electron microscope, operating at an accelerating voltage of 80 kV, in the ''Service d'Étude et de Recherche en Microscopie Électronique'' of the University of Corsica (Corte, France).

Vitellogenesis
The vitelline glands of C. metoecus contain vitellocytes at various stages of development, with younger cells localized in the periphery of the vitelline lobes. One cell type is observed and there are no cytoplasmic extensions between vitellocytes. Follicular vitellarium is surrounded by a basal lamina (Figs. 1, 3). Vitellocyte maturation is divided into four main stages. At the first stage, vitellocytes are undifferentiated cells showing a high nucleo-cytoplasmic ratio (Figs. 1, 15) and measure about 6 lm in diameter. The cytoplasm is mainly filled with free ribosomes, some mitochondria, and scarce endoplasmic reticulum sacculi (Fig. 2). Some heterochromatin is present in the nucleoplasm. At the second stage, the nucleocytoplasmic ratio decreases (Figs. 3,15), and Golgi complexes are evident in the cytoplasm (Fig. 4). At this stage, single shell inclusions coalesce to form clusters of electron-dense globules delimited by a membrane and measure about 10 lm in diameter. The nucleus often contains a large nucleolus (Fig. 3). At the third stage, single shell globules coalesce into clusters (Figs. 5,15) and vitellocytes measure about 14 lm in diameter. There are less mitochondria and reticulum endoplasmic sacculi (Fig. 6). Some small lipid droplets were seen in the cytoplasm, and glycogen particles were detected according to the Thiéry method ( Fig. 7). At the last stage, there are many shell globule clusters in the cytoplasm (>30 per cross-section), measuring about 19 lm in diameter. The cytoplasm also contains some mitochondria, and scarce endoplasmic reticulum sacculi at the periphery of the cell (Fig. 8). Mature vitellocytes of Crepidostomum metoecus possess a high amount of saturated lipid content (>10 lipid droplets per cell cross-section) (Figs. 8,9) and many glycogen particles (Figs. 10,15).

Oogenesis
The ovary is surrounded by a basal lamina (Fig. 11). It contains germ cells at various development stages that are closely packed, with younger cells generally localized in the periphery of the ovarian lobes (Fig. 11). Few organelles are observed between cells in the ovary. Four stages are observed during oogenesis in C. metoecus: oogonia, primary oocytes, developing oocytes, and mature oocytes. Oogonia are typically undifferentiated cells, showing a high nucleo-cytoplasmic ratio, measure about 5 lm in diameter and have a roundish shape. The scant cytoplasm is filled with free ribosomes and contains few mitochondria (Figs. 11,16); no nucleoli are present in the nucleus, only heterochromatin. Primary oocytes possess a higher nucleo-cytoplasmic ratio (Figs. 11,12,16) and measure about 8 lm in diameter. The cytoplasm contains chromatoid bodies in the perinuclear region and is filled with free ribosomes, mitochondria, and few granular endoplasmic reticula. The nucleus often shows a nucleolus at this stage. Developing oocytes (Figs. 11,13,16) enter in the zygotene-pachytene stage of the first meiotic division, as evidenced by the appearance of synaptonemal complexes in the nucleus (Fig. 13 inset) and measure about 11 lm in diameter. There may be up to eight synaptonemal complexes in a nucleus (by transversal section). The cytoplasm contains less mitochondrion but more granular endoplasmic reticulum. The mature oocytes are located in the central region of the ovary (Figs. 11,14,16) and possess elongated cytoplasmic extensions. These cells often show a triangular shape and measure about 18 lm in diameter. In all mature oocytes, a chromatoid body (2 lm of diameter) is present in the cytoplasm, near the granular endoplasmic reticulum and surrounded by mitochondria. Cortical granules are in a monolayer close to the periphery of the cell (approximately 50 per cell). The granular endoplasmic reticulum often takes a parallel shape, and mitochondria are clustered at a pole of the cell.

Vitellogenesis
Although vitellogenesis has a similar evolution in all neodermatan trematodes, some ultrastructural variations occur during maturation. Some vitelline follicles of Digenea possess interstitial cells (or nurse cells) with cytoplasmic extensions between vitellocytes that are believed to be involved in the selection and transport of materials from the parenchyma to the developing vitelline cells [1,13,14,28].
In Crepidostomum metoecus, as in Plagiorchis elegans, or Maritrema feliui, nurse cells were not found [15,46]. Vitelline cells provide nutrition for the eggs, and also provide the eggshell substance, in addition to the Mehlis' gland [13,28]. The abundance of endoplasmic reticulum, Golgi complexes,   and free ribosomes, produces this high synthetic activity [24]. This synthesis gives rise to shell globules that coalesce to form clusters of dense globules surrounded by a limiting membrane (polygranular content). The shape and amount of shell globules in the cluster, the number of clusters per cell, and the size of shell globules and clusters, differ by species (Table 1). Data listed in this table were either taken from the text of corresponding publications or obtained by measuring the structures from figures in these publications [15]. Vitelline cells accumulate nutritive reserves (lipid droplets and glycogen particles) for the developing embryo [30]. The nutritive amount varies among digenean lineages (Table 1). Thus two groups of trematodes are observed: those whose vitelline cells produce a large amount of nutritive reserves as is the case in C. metoecus [7,8,15], and those whose vitellocytes contain a small amount of nutritive substances [22,23]. Poddubnaya et al. (2013) investigated the vitellogenesis of Brandesia turgida and showed that the cytoarchitecture of vitellocytes reveals a specific life cycle. According to their analysis, the development of C. metoecus larva seems to occur after egglaying, due to the high quantity of nutritive reserves in vitellocytes, unlike Brandesia turgida, which possess few nutritive substances [38]. The presence of such substances in eggs is necessary for the developing embryo that is less able or not able to receive nutrients from the parent worm. In vitelline cells of C. metoecus, only b-glycogen particles have been observed, as in other species [24,30,37,42]. In some trematodes, a-glycogen rosettes and b-glycogen particles have been found [15]. The lack of densely coiled endoplasmic reticulum in fully mature vitellocytes of C. metoecus contributes to the conclusion that maturation ended at the fourth stage. In some other digenean species, a fifth stage presented secretion of densely coiled endoplasmic reticulum [12,15,30] and glycogen particles that are condensed in the whole cytoplasm. Moreover, vitelline cells providing shell globules participate in eggshell formation to protect the developed embryo. In the present study, we observed a great amount of shell globule clusters (about 30 per cell cross-section) composed of many shell globules (>30 globules/cluster). Nevertheless, the cluster size is one of the lowest in the vitelline gland of digenean species (Table 1). The variation of eggshell supply can be explained by the difference of life cycle between trematodes [1]. This feature can represent a phylogenetic point of comparison between families of trematodes, like many other characteristics.

Oogenesis
On the basis of the female gonad structure, Platyhelminthes are subdivided into two levels of organization. In Neoophoran Platyhelminthes, the production of yolk and shell-forming precursors occurs in vitellaria, unlike in Archoophorans where it takes place in germaria [9]. The presence of organelles between growing oocytes supposes the presence of interstitial cells that may transport organelles. The initial phase of oocyte maturation takes place during the prophase of the first meiotic division in the ovary. As for Metadena depressa [14], no nucleus has been observed in the thin interstitial cytoplasmic layer between growing oocytes and the basal lamina, unlike in other Platyhelminthes where syncytial structures may occur in the ovary [34]. Oogonia are present along the wall of the ovary as for Cryptocotyle lingua [5], but do not contain a nucleolus-like Zygocotyle lunata [49]. Developing oocytes enter the zygotene-pachytene stage of the first meiotic division recognizable by the presence of synaptonemal complexes in the nucleoplasm, except in some digenean species where it takes place in the uterus [18]. During maturation, oocytes migrate to the center of the ovary. The metaphase takes place in the proximal part of the uterus where fertilization occurs [17]. A granular mass transfer from nucleus to cytoplasm occurs before the last stages of maturation. This material, named either ''chromatoid body'', ''nuclear extrusion'', or ''nucleolus-like cytoplasmic body'' [4,18,27,35], probably contains RNA [4,25]. This structure is often surrounded by mitochondria and granular endoplasmic reticulum [14], as in the present study. Movements of mitochondria during oogenesis were described by Yosufzai (1952), who studied this in Fasciola hepatica, where mitochondria are at one pole of oogonia, then form a ring around the nucleus in growing oocytes, and are finally evenly distributed in the cytoplasm of mature oocytes [52]. Such movement has not been observed in C. metoecus. In the fully mature oocytes, inclusions are obvious near the cytoplasmic membrane. These small vesicles initially randomly distributed in the cytoplasm, migrate to the cortical cytoplasm where they form a monolayer just beneath the oolemma. This can be observed in several fully mature digenean oocytes [2,4,6,11,18,19,27,31,34,36,49,51]. The function of these granules could be the same as that of oocytes of other animals, which act in the blocking of polyspermy during fertilization [31]. The Thiéry method [47] allows us to exclude protein composition of these particles. The low protein composition of mature oocytes differs from that of other studied trematodes, such as Paramphistomum cervi [20], Gastrothylax crumenifer, and Ceylonocotyle dawesi [26]. This pattern, associated with the large nutrient content of vitellocytes of C. metoecus, enables us to consider that oocytes do not take part in the nutrition of the future embryo of the miracidium. Although some minimal differences occur between oogenesis of C. metoecus and some other digeneans, the general pattern of oogenesis corresponds to other neodermatan worms, and more generally to Platyhelminthes.