Incipient spontaneous granulosa cell tumour in the gerbil, Meriones unguiculatus M A Guzma´n-Silva and M Costa-Neves Department of Pathology, Federal Fluminense University, Niteroi, RJ, Brazil

Summary In 167 cases of granulosa cell tumour (GCT) in the gerbil, Meriones unguiculatus, 42.5% (71 cases) were in the ovaries, with no evidence of macroscopic lesion. In this paper, we describe the common findings in the incipient – microscopic – type of this spontaneous ovarian tumour occurring in gerbils younger than two years of age. Keywords Gerbil; granulosa cell tumour; ovary

The granulosa cell tumour (GCT), one of the tumours of the theca-granulosa cells, is one of a group of neoplasms derived from the strome of the sexual cord (Crum 2000, Miranda et al. 2000). These tumours originate from the ovarian mesenchyme and have clinical importance, once they synthesize great amounts of oestrogens, or they can have malignant effects (Crum 2000, Miranda et al. 2000). In addition, it was observed that, in specific cells of ovary carcinomas, they can produce a great quantity of steroid hormones, which explains the virilizing effect of certain GCTs (Crum 2000). As the spontaneous GCT in the gerbil reproduces in some respects the human ovarian neoplasm, its use as a model of the disease allows the study of several aspects, such as tumour growth and progression, development and metastasis pattern, therapeutic procedures, and hormone and immune modulation. The aim of this study was to define the biological characteristics – incidence, macroscopy, histopathology and metastases – of the incipient type of GCT in the gerbil. Correspondence: Maria Angelica Guzman-Silva, Rua Pereira Da Silva 137/1806, 24220-030 Icarai, Niteroi, RJ, Brazil. Email: [email protected] Accepted 21 April 2005

Materials and methods Seventy-one cases of incipient GCT in gerbils were analysed, without any macroscopic alteration of the ovaries. The animals were maintained in the Animal Facility of the Section of Experimental Pathology in the Department of Pathology at the Federal Fluminense University. All the animals received commercial food and filtered water ad libitum. Animals were monitored five days a week, and no signs of sickness, discomfort or distress were noticed. The gerbils were classified into virgins and reproducers and distributed in three age groups for the analysis. After complete necropsy, fragments of several organs were collected besides both ovaries, fixed in 10% buffered formalin, and processed for embedding in paraffin, as is usual in histopathology; 5 mm sections were stained with haematoxylin and eosin. Besides the microscopic characteristics of incipient GCT, the presence, frequency and metastases location were analysed, defining whether the GCT was malign or not. The testosterone level was analysed in 22 cases of bilateral incipient GCT. The blood was collected from the retro-orbital sinus vein, with the animals properly

r Laboratory Animals Ltd. Laboratory Animals (2006) 40, 96–101

Incipient granulosa cell tumour in the gerbil

anaesthetized by ether inhalation before being killed through cervical dislocation. The serum was stored at 201C for the androgen test by the solid phase radioimmunoassay technique (Samanta & Ali 1990). Serum obtained from normal males and females was used as a control.

Results Forty-six incipient tumours were bilateral, representing 64.8% of the total incipient GCTs. Unilateral incipient GCT occurred in the right ovary (RO) in 17 cases (23.9%) and in the left ovary (LO) in eight cases (11.3%). The frequency by sexual status was 44 in virgins (62%) and 27 in reproducers (38%). The age group with the larger incidence of incipient GCT was the one of 1–2 years, with 36 cases (50.7%). Virgins from 1–2 years acccounted for 34 cases (47.9%) and reproducers from 2–3 years for 17 cases (23.9%) (Figure 1). Microscopically, GCT was characterized as unilateral incipient lesions in 25 cases (35.2%) and bilateral incipient lesions in 46 cases (64.8%), giving a total of 117 tumours (Figure 2). The tumours were mainly composed of nests and cords of cubical tumour cells, some were observed in the cystic wall and others substituted the germinative epithelium (Figure 3A). The other findings were luteinic cells (RO 56; LO 47) (Figure 3B), incipient cysts (RO 50; LO 41), Call–Exner bodies (RO 16; LO 12)

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Reproducer

35

Virgin

30 n

25

97

Precocious tumour lesion Cysts Call-Exner bodies Pseudofollicles Luteinic cells

Right ovary Left ovary

0

20 40 60 80 100 120 n

Figure 2 Microscopic characteristics of 117 incipient granulosa cell tumours of gerbils distributed by anatomic location

and pseudofollicles (RO 4; LO 3) (Figure 3C). The frequency of luteinic cells varied from low to high, and a high number was present in most of the cases. Some cysts were already well developed. Mitosis was not observed. Local invasion was detected in the ovarian hilus in 54 cases (RO 49; LO 34) (Figure 3D), periovarian fat in 15 cases (RO 15; LO 11), fimbriae in 17 cases (RO 13; LO 11) (Figure 3E) and large ligaments in three cases (Figure 4). Metastases occurred in the abdomen, the omentum being the most affected organ (30 cases) (Figure 3F), followed by the mesentery (three cases). Metastases were not found in the thorax. Serum testosterone was determined in 14 virgins and eight reproducers with bilateral incipient GCT, and the values varied between 0 and 4.80 ng/mL. In the virgins, the mean value was 1.30 ng/mL and, in the reproducers, it was 0.40 ng/mL. When the values were analysed by age, it was found that the average for the group of 1–2 years was 1.30 ng/mL, 0.35 ng/ml for the group of 2–3 years and 0.70 in the older group. No statistical difference was detected (Mann–Whitney test).

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Discussion

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Our data on this neoplasm were obtained through systematic necropsy and exhaustive investigation of gerbils from the stock of our colony. Spontaneous ovarian and other tumours can appear in gerbils from two years of age (Benitz & Kramer Jr 1965, Rowe et al. 1974,

5 0 1–2 years

2–3 years

> 3 years

Age Figure 1 Distribution by age and sexual status of 71 incipient granulosa cell tumour in gerbils

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Figure 3 Incipient granulosa cell tumour of the gerbil (haematoxylin and eosin stain). (A) Tumour nest of cuboidal cells substituting the ovarian germinative epithelium. (B) Tumour nest and cords of luteinic cells. (C) Call–Exner body (arrow) and pseudofollicles. (D) Tumour invasion of the ovarian helium. (E) Tumour invasion of the de fimbriae. (F) Incipient granulosa cell tumour metastases in the omentum

Vincent & Ash 1978, Meckley & Zwicker 1979, Vincent et al. 1979, Guzma´n-Silva & Rossi 1990, Guzma´n-Silva et al. 1995a, Matsuoka & Suzuki 1995). In this sampling, we verified the occurrence of GCT in gerbils Laboratory Animals (2006) 40

aged less than two years. Most of the incipient tumours were bilateral, representing 71 (64.8%) of the total, 44 of them in virgins (62%) and 27 in reproducers (38%). Incipient GCT can appear precociously in

Incipient granulosa cell tumour in the gerbil

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Indeterminate Left ovary Right ovary

Large ligament Fimbriae Periovarian fat Ovarian hilus 0

10

20

30

40

50

n Figure 4 Distribution by anatomic location of local invasion of 71 cases of incipient granulosa cell tumour in gerbils

gerbils of 1–2 years old. As the animal grows, these cystic and solid tumours can develop (dates not presented). The aetiology of GCT is still unclear. Epidemiological data indicate an increased incidence of ovarian epithelial cancer, with early menarche, late menopause and nulliparity. GCT is both regulated by and can synthesize hormones; it therefore seems logical that the molecular changes which occur in a granulosa cell and lead to its neoplastic transformation involve these endocrine pathways (Fuller & Chu 2004). The higher incidence of GCT in virgins could be explained by pregnancy protecting against GCT development, in rodents as well as in humans, since it causes a break in the cyclical action of FSH. Microscopically, gerbil GCT is composed of cubical cells in diffuse arrangement, in nests and cords, cysts, or with a follicular pattern, as well as mitosis (Benitz & Kramer Jr 1965, Ringler et al. 1972, Meckley & Zwicker 1979). The same features are observed in human GCT (Kurman et al. 1979, Chadha et al. 1990, Haba et al. 1993). Haemorrhagic cysts of several sizes have been described in the GCT of gerbils (Meckley & Zwicker 1979) and mice (Beamer et al. 1985). A prevalence of vacuolated luteinic cells has been described in gerbils (Benitz & Kramer Jr 1965, Ringler et al. 1972, Rowe et al. 1974). Call–Exner bodies have only been described in human GCT (Kurman et al. 1979, Chadha et al. 1990), but we have identified them in gerbil GCT. The microscopic aspects of the

incipient tumours were characterized by tumour cells in nests and cords; some of which were observed in the cystic wall, and with others substituting the germinative epithelium. Luteinic cells and incipient cysts were observed as well as Call–Exner bodies. Mitosis was not observed, probably due to a low proliferative rate during the precocious phase of the tumour development. The gerbil ovary presented histopathological alterations of GCT, mainly related to luteinization of the tumour cells, even if no ovarian alteration was observed macroscopically. Therefore, the study of precocious lesions of this tumour is very important, as they can result in significant hormonal alterations. Long-term studies in this species should be attentive to the interference of this pathology. Only one reference describes the invasion of GCT in the uterine serosa and myometrium of gerbils (Rowe et al. 1974). Local invasion of incipient GCT was most frequent in the ovarian hilus (54 cases), followed by periovarian fat and fimbriae. The largest number of metastases occurred in the abdominal omentum (30 cases). Our data agree with that described in mice (Beamer et al. 1985). Only one reference tells about the absence of metastases in GCT of gerbils (Meckley & Zwicker 1979). Metastases of GCT have already been observed in abdominal organs such as lymph nodes, kidneys, pancreas, liver and diaphragm (Beamer et al. 1985). Considering the local invasion and metastases, most of the incipient GCT in gerbils (84.5%) was classified as malignant tumours. Only four bilateral and seven unilateral GCT did not show malignant behaviour by microscopy. In the incipient GCT, the serological level of testosterone varied between 0 and 4.80 ng/mL in 22 cases, and the average was 1.80 ng/mL. By sexual status and age, the average was higher in virgins of less than two years old (1.30 ng/mL). In normal gerbils, using the same methodology for the detection of serological testosterone, we found an average of 0.13 ng/mL in females and 0.82 ng/mL in males (Guzma´n-Silva Laboratory Animals (2006) 40

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et al. 1995b). Since the mean value found in females carrying incipient GCT was higher than that in normal males, it seems that a high level of testosterone could be related to GCT development in gerbils. Androgens acting synergistically to FSH interfere in the lipoprotein metabolism in ovarian granulosa cells (Schreiber et al. 1984). The same synergism could play a role in the regulation of proliferation in those cells, which would have some contribution to tumorigenesis. In human GCT, the immunoreactivity of oestradiol and progesterone has been detected more constantly and the results related to testosterone have already been recognized (Kurman et al. 1979, Chadha et al. 1990, Mulvany & Riley 1997). In humans, testosterone production by GCT cells is associated with several degrees of virilization (Kurman et al. 1979, Chadha et al. 1990). In the gerbil GCT secreting androgens can apparently present a virilizing behaviour. The histopathological findings of gerbil GCT resemble the ones observed in the GCT of rodents and humans. The initial spreading of the tumour begins in the peritoneal cavity, and metastases probably occur in the abdominal organs due to lymphatic and haematogenous spread. Incipient GCT in gerbils occurs in animals of less than two years of age, mainly in virgins. This precocious tumour is similar to that of the human ovarian neoplasm, and can be used as an animal model of the disease. It would therefore facilitate future studies of genetic, hormonal and immune modulation, and the action of antineoplasmic drugs during the whole period of the ovarian tumour development. Acknowledgement The authors are grateful to the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) for the support during this research.

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