Bull. Eur. Ass. Fish Pathol., 32(1) 2012, 19

* Corresponding author’s e-mail: luciana.mandrioli@unibo.it

NOTE

Ultrastructural findings of spontaneously occurring seminoma in an adult ornamental

cyprinid (Cyprinus carpio L.)

R. Sirri1, C. Brachelente2, H. Schmidt-Posthaus3, G. Vitellozzi2 and L. Mandrioli1*

1 Department of Veterinary Medical Sciences, Service of Anatomic Pathology, Via Tolara di Sopra 50, 40064, Ozzano Emilia, Bologna, Italy; 2 Department of Biopathological Sciences and Hygiene of Animal and Food Productions, Via San Costanzo, 4, 06126 Perugia, Italy; 3 Centre for Fish and Wildlife Health,

Institute of Animal Pathology, University of Bern, Laenggassstrasse 122, 3012 Bern, Switzerland

Abstract An ultrastructural description of a previously described spontaneous seminoma in a koi carp (Cyprinus carpio) is given in this note. The seminoma, diagnosed as classical with a diffuse pa�ern by histology and immunohistochemistry, displayed a homeomorphic population of neoplastic cells with ultrastructural features similar to those described in human seminoma.

Histopathological and immunohistochemical findings of a seminoma in a three-year-old, male, koi carp has been recently reported (Sirri et al., 2010). The animal presented a symmetric abdominal enlargement; at necropsy a celomatic multinodular, white-yellowish and firm mass infiltrating the liver and the intestinal wall was detected. Histologically, the neoplasm was non-encapsulated and poorly demarcated, showed invasive growth and was characterized by a lobular architecture, subdivided by abundant fi-bro-connective septae (Figure 1a). Large necrotic and calcified areas in association with small aggregates of residual spermatids were present (Sirri et al., 2010). An immunohistochemical phenotypization of the tumour was performed

to exclude other celomatic neoplasms. Neo-plastic cells cross-reacted with markers of the seminomatous cells (vimentin, placental alka-line phosphatase -PLAP, and c-KIT) (Sirri et al., 2010). A diagnosis of a classical seminoma with a diffuse pa�ern has been formulated.

Here we describe the ultrastructural features of this seminoma to further characterize the mor-phology of the seminomatous cells. Immedi-ately a�er death, portions of the neoplastic mass were formalin-fixed and routinely processed for histopathology; electron microscopy was performed subsequently from formalin-fixed samples because, although not optimal, experi-ence in our laboratories with other tissues had

20, Bull. Eur. Ass. Fish Pathol., 32(1) 2012

demonstrated a confident structural preserva-tion in formalin-fixed material; samples were then dehydrated in a graded alcohols sequence, and embedded in Durcupan AcM resin. Semi-thin sections were stained with toluidine blue and examined by light microscopy. Selected ultrathin sections were cut and stained with uranyl acetate and lead citrate. The ultrastruc-tural observation was made with a Philips TEM 208 transmission electron microscope (Philips, Eindhoven, Holland) operating at 100 kV.

Seminomatous cells displayed a monomorphic population (Figure 1b, 2a). At higher magnifi-cation, fragmented nucleolus and dispersed chromatin were present (Figure 2b). Nuclear atypia and invagination of the nuclear mem-brane (arrow) were o�en detected (Figure 2c). Seminomatous cells had swollen mitochondria (Figure 2d, arrow). Intercellular junctions were not detectable, as well as glycogen rose�es. The ultrastructural examination revealed some features of the seminomatous cells (dispersed chromatin, nucleolar fragmentation, nuclear

membrane invagination), which are also re-ported in seminomas of human beings (Hend-erson et al., 1986), reinforcing the histological diagnosis, while other ultrastructural features characteristic of seminoma as the ribbon-like nucleolonema were not detected (Henderson et al., 1986). In addition, the swollen mitochondria of the seminomatous cells seen at the ultrastruc-tural examination have been interpreted as a sign of cell degeneration. Even if ultrastructural descriptions of seminoma in other species are rare, the findings reported are similar includ-ing human beings (Brown and Stafford, 1989; Doster et al., 1989; Saied et al., 2011).

The ultrastructural examination is a valid ap-proach to study the morphology of sponta-neous tumours, becoming an indispensable requisite in case of anaplastic or undifferenti-ated tumours, occurring in fish species (Kagan, 2010). In mammals and man, this method is a valuable tool in the investigation of possible aetiologic agents (especially viral particles) that have been proven to be related to the occurrence

Figure 1. Koi carp (Cyprinus carpio). Seminoma. (a) Seminoma is characterized by a lobular architecture, subdivided by abundant fibro-connective septae. (bar=200 μm) (H&E). (b) Semi-thin section of seminoma showing a monomorphic cell population characterized by frequent nuclear atypia (bar=25 μm) (Toluidine blu).

Bull. Eur. Ass. Fish Pathol., 32(1) 2012, 21

of a tumour (i.e. retrovirus for walleye dermal sarcoma) (Martineau et al., 1990); it also gives a detailed description of morphological changes that may occur in different various cellular compartments a�er tumour transformation.

Several papers focused on the use of transmis-sion electron microscopy (TEM) in spontaneous fish tumours: cutaneous papilloma in Atlantic salmon (Salmo salar) (Carlisle et al., 1977) and orocutaneous papilloma in brown bullheads (Ictalurus nebulosus) (Poulet and Spitsbergen, 1996), cutaneous lymphoma in northern pike (Esox lucius) (Thompson and Mie�inen, 1988),

dermal sarcoma of walleye (Stizostedion vitreum) (Martineau et al., 1990), pancreatic adenoma and adenocarcinoma in mummichog (Fundulus heteroclitus) (Vogelbein and Fournie, 1994), hepa-toblastoma in mummichog (Fundulus heterocli-tus) (Vogelbein et al., 1999), dermal melanoma in brown bullhead catfish (Ictalurus nebulosus) (Sakamoto and White, 2002), squamous cell carcinoma in rudd (Scardinius erythrophtalmus) (Hanjavanit and Mulcahy, 2004), neuroectoder-mal tumors and retinoblastoma in various fish species (Kagan, 2010).

Figure 2. Koi carp (Cyprinus carpio). Seminoma, TEM. (a) Seminoma cells display a quite homeomorphic population (bar=10 μm). (b) A seminomatus cell displays fragmented nucleolus and dispersed chromatin (bar=2 μm). (c) Nuclear atypia and invagination of the nuclear membrane (arrow) (bar=2 μm). Collagen fibers (*) are present in the stromal component. (d) A seminomatous cell displays swollen mitochondria (arrow) (bar=2 μm).

22, Bull. Eur. Ass. Fish Pathol., 32(1) 2012

TEM has also been used for the discrimination between neurofibromas and schwannomas in goldfish (Duncan and Harkin, 1969), a difficult diagnosis that can be achieved with this specific approach. Concerning the fixation methods, al-though glutaraldehyde is suggested for optimal tissue preservation, formaldehyde-fixed mate-rial (as in case of archived samples) is o�en acceptable and the superiority of buffered for-malin as an EM fixative compared to unbuffered formol-saline is strongly emphasized (Holt and Hicks, 1961; Henderson et al., 1986).

In conclusion, with this note, a suggestion to include TEM procedure in the classic diagnostic algorithm is highly advisable; the interpretation per se of the morphological findings depends, however, on a comparative study of the images obtained. Moreover, further studies are needed on healthy tissues as well, since references about TEM features in different fish species and organs are scarce.

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