Neoplasia of the Sertoli Cells in Wild Carp, Cyprinus Carpio

84, Bull. Eur. Ass. Fish Pathol., 33(3) 2013

Neoplasia of the Sertoli cells in wild carp, ¢ȱ: optical, immunohistochemical and ultrastructural study

L. A. Romano1Șǰȱǯȱ ě2, F. Fuhr, R. V. Rodrigues2, E. Pereira Gusmao2, M. A. R. Garrido- Pereira2, L. A. Sampaio3 and M. B. Tesser3

1 ȱȱęǰȱȱȱȱȱ ȱàȱȱ ȱȱ ȱȱȱǲȱ2ȱȱàȬ ³¨ȱȱǰȱȱ ȱȱȱ ǰȱàȱȱȱȱǰȱȱşŜŘŖŗȬşŖŖǰȱ ȱ ǰȱǰȱ£ǲȱř ȱȱęǰȱȱȱȱȱ ǰȱ àȱȱȱȱȱǰȱȱşŜŘŗŖȬŖřŖǰȱȱ ǰȱǰȱ£

Abstract Sertoli cell tumours are primary neoplasms of the testis which arise from the supporting cells within the seminiferous tubules. This report describes a case of tumour of the Sertoli cells in carp. The diagnosis of Sertoli cell tumour is supported by the histopathological features, the characteristic ȱȱȬãĴȱ¢ȱȱȱȱȱȱ¢ȱȱ¢ȱ ȱȱȱȱȱȱęȱȱǻǼȱȱȱ΅ǯ

Introduction tumours in the higher vertebrates, mammals ȱęȱȱȱȱȱȱ ȱǲȱȱȱĴȱȱȱȱ diverse class of vertebrates, with over 20,000 ȱȱ ȱǰȱęǰȱȱȱ known species. Their diversity and placement in ȱȱȱęȱȱęȱȱ the phylogenetic tree make them ideal subjects is consequently largely based on mammalian for comparative neoplasia studies. Fish share criteria (Romano, 1987; Masahito, 1998). a common histiogenesis of various neoplasms ȱȱȱǰȱ ȱȱĴȱ All the major varieties of neoplasias that occur understanding of the basic mechanisms of car- in mammals and birds, have been recorded in cinogenesis, compared with studies limited ęǯȱȱȱȱȱȱǰȱ to mammalian models only. (Anders, 1984; mesenchymal tissues, lymphoid tissue, nervous Schmale, 1986; Baumann et al., 1991; Webe et tissues, haematomas and melanomas (Romano, al., 2002; Devi, 2010). 2004 ; Romano, 2010)

Previously such studies have dealt largely with ȱǰȱȱęǰȱȱȱǰȱȱȱ

* Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 33(3) 2013, 85 number of supportive functions in regard to rehydrated with. The endogenous peroxidase the spermatogenic maturation process, certain activity was blocked by incubating the slides changes and proliferation of these cells as an for 20 min in 0.3% H2O2 in a 5% methanol so- been described (Blazer, 2002). Sertoli cell neo- lution. After washing the slides in water and plasia is a primary neoplasm of the testis arising ȦŖǯŖśƖȬ ȱŘŖȱǰȱ¢ȱ ȱ- from the supporting cells within the seminifer- ȱȱȱŗȦŗŖŖȱȱǻȱ ous tubules. It is common in dogs, especially in Universal Elite, BC Kit, Vector), in a 10% PBS cryptorchid testicles, and has also been reported bovine serum albumin (BSA) solution for 30 in the stallion, ram, cat, and bull (MacLachlan min at 22 °C in a humid chamber. After incuba- and Kennedy, 2002). Normal and neoplastic ǰȱȱ¢ȱȬȱ¢ǰȱȱ΅ǰȱ Sertoli cells are reported to be the only cells of ȱȬęȱȱǻǼȱ¢ȱ the testes that stain immunohistochemically (Dako, Argentina) and the slides were incubated ȱ¢ȱȱȬęȱȱǻǼȱ overnight at 41°C in a humid chamber. The (Owston and Ramos-Vara, 2007; Saegusa et slides were then rinsed in PBS and incubated al., 2011). They also stain with vimentin, and for 7 min in a 50 mL 30.3-diaminobenzidine there are varied reports of staining with other (DAB, Sigma- Aldrich) solution containing 1% antibodies including S100, melan-A, and inhibin PBS-BSA in 50 mL H2O2. Counterstaining was ΅ǯȱ¢ȱȱ ȱȱǻȱ then performed with haematoxylin. ȱǯǰȱŘŖŗŘǼǯȱȱȱ΅ȱȱȱ¢ȱȱ Sertoli cell neoplasia in canine and human and For electron microscopy small fragments of ȱȱȱȱęȱȱȱȱ the tumour were cut into 1 mm blocks and neoplasm (Kuroda et al., 2004) ¢ȱę¡ȱȱȱěȱ- taraldehyde (pH 6.9 at 4 °C), washed in Mil- Materials and methods ȂȱȱȱȬę¡ȱȱŗƖȱȱ A mass of 23 g was obtained from the abdominal tetroxide; the tissue blocks were then dehy- cavity of a carp from the river Salado, Buenos drated in a graded series of ethanol-acetone, ǰȱǯȱȱęȱȱȱ ȱȱŗřŜŝȱȱ immersed in propylene oxide and embedded and a total length of 620 mm. Necropsy showed in Durcupan ACNI (Fluka Chemie A.G., Swit- a tumour of 22 cm diameter found in the ab- zerland). Ultrathin sections were cut with an dominal cavity. The tumour was whitish and LKH ultramicrotome and doublestained with solid lobed with haemorrhagic areas (Figure uranyl acetate and lead citrate before examina- ŗǼǯȱȱȱȱȱ ȱę¡ȱȱŗŖƖȱ tion in a Jeol JEM-8T electron microscope (Jeol, ěȱǰȱȱȱĜȱȱ Tokyo, Japan). stained with haematoxylin and eosin, PAS and oil red O (for lipid staining) on frozen sections. Results Histological sections were stained with im- Neoplasic cells were oval or round contained munohistochemical procedures, according to round nuclei and abundant vacuolated cyto- ȱęȱȬ¡ȱ¡ȱ plasm, and was arranged in nested and cord- technique (Hsu, 1981). Tissue slides were de- ȱĴȱȱ¢ȱȱȱ Ĝ£ȱȱȱ ȱ¡¢ȱȱȱ ȱ¢¢ȱęȱǻȱŘǼǯȱȱ 86, Bull. Eur. Ass. Fish Pathol., 33(3) 2013

Figure 1. Gross appearance of the abdominal Figure 3. The tumour cells were oval or round tumor with multiple masses of various sizes. contained round nuclei and abundant vacuolated (arrow), Bar= 2 cm. cytoplasm, and was arranged in nested and cord- ȱĴȱȱ¢ȱȱǯȱ ¢¢ȱęȱȱȱǻ Ǽȱ ǭȬ ǰȱȱƽȱśŖȱΐǯ

Figure 2. Neoplastic cells are arranged in tubules irregularly distributed tending to position perpendicular to the basement membrane (arrow). Between the tubules are observed lymphocyte Figure 4. Tumour mass with hyaline areas (H) and ęȱǻȘǼǯȱ ǭǰȱȱƽȱŗŖŖȱΐǯ ȱ ȱȱęȱǻ Ǽǯȱ ǭȬǰȱ ȱƽȱśŖȱΐǯ

ęȱ ȱ¢ȱȱȱȱ¢ȱȱ Ultrastructural examination revealed a pseu- ŖȮřȱȦȬ ȱęȱǻȱřǰȱŚǼǯȱȱ dostratilied disposition of the nuclei. In the tumour cells contained pigments which were supranuclear cytoplasm, a well developed stained red by oil red O staining, indicating that Golgi complex, small vesicles, and parallel cis- ȱȱȱĴ¢ȱȱǻȱśǼǯ ternae of rough endoplasmic reticulum were Immunohistochemical examination revealed the most prominant features (Figure 8). In the that the tumour cells were positive for NSE most immature cells was observed in the cyto- ȱȱ΅ȱȱ ȱȱȱ plasm moderate development of both rough cytokeratin antibody. (Figure 6, 7). and smooth endoplasmic reticulum, as well Bull. Eur. Ass. Fish Pathol., 33(3) 2013, 87

Figure 7. Immunohistochemical of the Sertoli cells. Figure 5. Tumour cells with pigments were The tumour cells show cytoplasmic membrane stained red by oil red O staining, indicating that ȱ ȱ ȱ ȱ ΅ǯȱ Ȭȱ ȱ ΅ȱ ȱȱȱĴ¢ȱǯȱȱȱȱ ǰȱȱƽȱśŖȱΐǯ ǰȱȱƽȱśŖȱΐǯ

Figure 6. Immunohistochemical of the Sertoli cells. The tumour cells show cytoplasmic and nuclear- positive staining for NSE. Anti-NSE staining, Bar ƽȱśŖȱΐǯ as of the Golgi complex, and the presence of ȱȱȱȱęǯȱ ȱȱȱǰȱ¢ȱȬãĴȱ crystals were also present (Figure 9) and lipid inclusions were present in most neoplastic cells examined. The nucleus generally showed deep infolding. These cells were observed in isola- tion and are similar to human Type C or nearly Figure 8. Neoplastic Sertoli cells. The nuclei are mature Sertoli cell found in the Del Castillo’s round to eleongated and regular in outline and ȱȱęȱǯȱȱƽȱŘȱΐǯ syndrome (Paniagua, et al., 1987). The nucleolus was more prominent than in mature Sertoli cells (Figure 10). 88, Bull. Eur. Ass. Fish Pathol., 33(3) 2013

Figure 10. Immature Sertoli cell. The nucleus shows a deep infolding (arrow). The nucleolus is more prominent than in mature Sertoli cells. Bar ƽȱŖǯŗȱΐǯ

adaptation is the ability to detoxify and repair damage caused by toxins that may be carcino- genic (Stoskopf, 1993). In studies of hybrid carp. Figure 9. Immature Sertoli cells. The cytoplasm from a eutrophic lake of Spain, gonadal tumors shows moderate development of the endoplasmic ǰȱ ȱ ęȱ Ȭ were found only in males (Granado-Lorencio, ãĴȱ¢ȱǻ ǼǯȱȱƽȱŘȱΐǯ et al., 1987). The most prevalent type was dys- germinoma; other tumors, of lower prevalences, included seminomas, leiomyomas, Sertoli cell tumors and spermatocytic seminomas Discussion Neoplasia processes are ubiquitous among Two types of testicular neoplasia were evident vertebrates, and comparison has shown that ȱ¢ȱęȱȱȱȱǯȱ neoplasias in cold-blooded animals are essen- Both types of neoplasias appeared to be of tially identical in regard to their structure and Sertoli cell origin. One contained predomi- behavior with the corresponding neoplasias ¢ȱȱȱ ȱĴȱȱȱȱȱ of warm-blooded animals. Neoplastic disease and germinal cells in only small numbers. The in carp is well represented in the literature, second contained large numbers of spermato- ¢ȱěȱęǯȱȱȱ gonia together with Sertoli cells that contained ȱȱȱȱȱęȱȱȱ¢ǰȱ large amounts of lipid and smooth endoplasmic ¢ȱȱǯȱ ęȱȱ ȱȱ ȱ reticulum. Small, apparently inactive Leydig adapted to environments that are suboptimal cells were present in both types of neoplasias ȱȱȱȱęǯȱȱȱȱȱ ȱȱȱȱȱęȱȱȱȱ Bull. Eur. Ass. Fish Pathol., 33(3) 2013, 89

brown bullhead ( ȱ) and neoplasias (Leatherland and Sonstegard, 1978). sediment contaminants in tributaries of the Laurentian Great Lakes. Canadian Journal of It is well known that the Sertoli cell is the most ȱȱȱ 48,1804-1810 resistant cell of the seminiferous epithelium. Blazer VS (2002). Histopathological assessment (Clermont and Perey, 1957; Heller et al., 1968), of gonadal tissue in wild fishesȱȱ In addition, it has been shown that this cell ¢¢ȱȱ¢ȱ26, ŞśȮŗŖŗǰ does not undergo mitotic division in the human Clermont Y and Perey B (1957). Quantitative adult testis (Steinberger and Steinberger, 1976). study of the cell population of the seminiferous tubules in immature rats. Because of this stability, the use of the Sertoli American Journal of Anatomy 100, 241-268. cell as a reference cell or constant compensat- Devi PU (2010). Basics of carcinogenesis. Health ing factor for the quantitation of the germi- Administrator 17, 16-24. ȱȱȱěȱ¢ȱȱ Granado-Lorencio C, Francisco Garcia-Novo pathologic conditions has been proposed (Nistal F and Lopez-Campos J (1987). Testicular et al., 1982). tumors in carp-funa hybrid: Annual cycle ȱěȱȱȱ ȱǯȱJournal of The distinctive histologic and immunohisto- ȱȱ23, 422-427 chemical features of the testicular neoplasias Heller GV, O’Keefe KB and Heller I C (1968). and the lack of any continuity between them ěȱȱȱȱȱǻ Ǽȱ on Sertoli cells in the hypophysectomized ȱȱȱȱ¡ȱȱȮ¡ȱȱ rat. Clinical Research 98, 113-123. ȱȱȱȱěȱ- Hsu SM, Raine L and Fanger H (1981). Use sis. The immunohistochemical expression of the of avidin-biotin-peroxidase complex Sertoli cell neoplasia is similar to that reported (ABC) in immunoperoxidase techniques: for NSE in normal and neoplastic Sertoli cells a comparison between ABC an unlabeled ȱȱǻȱȱǯǰȱŗşşŖǼǯȱȱȱ΅ȱȱ antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry 29, 577-585. reported only in Sertoli cell neoplasia in canine ȱȱȱȱȱȱȱęȱ Kuroda N, Senzaki T, Yamasaki Y, Miyazaki E, Hayashi Y, Toi M, Hiroi M, Enzan H marker for this neoplasm (Kuroda et al., 2004). (2004). Sertoli cell tumor of the testis (not The diagnosis of Sertoli cell neoplasia in the ȱęǼȱ ȱȱ¡ȱ present case is supported by the morphologi- of neuroendocrine markers and without cal and staining characteristics, ultrastructure ȱ ¡ȱ ȱ Ȭ΅ǯȱ Pathology International 54ǰȱŝŗşȮŝŘŚǯ ȱ¢ȱȱȱȱȱ΅ȱǯ Leatherland JF and Sonstegard RA (1978). References Structure of normal testis and testicular tumors in cyprinids from Lake Ontario. Anders F, Schartl M, Barnekow A and Anders Cancer Research 38, 3164-3173. A (1984). as an in vivomodel for studies on normal and defective control MacLachlan N J and Kennedy P C (2002). of oncogenes. Advances Cancer Researcher Tumors of the genital systems. In: Tumors 42, 191-275. in domestic animals 4th ed (D. J. Meuten, Ed.). Iowa State Press, Ames, Iowa pp. Baumann PC, Mac MJ, Smith SB and śŚŝȮśŝřǯȱ ǱȱşŝŞȬŖȬŞŗřŞȬŘŜśŘȬřǯ Harshbarger JC (1991). Tumor frequencies in walleye (£ȱ ) and Masahito P, Ishikawa T and Sugano H (1998). 90, Bull. Eur. Ass. Fish Pathol., 33(3) 2013

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