Sex cord-stromal (granulosa cell) tumor in an ovotestis from a cow

Abstract

Development of gonadal tumors within an ovotestis is rare in mammals and this disturbance was not reported in cows. We report herein a gonadal stromal (granulosa cell) tumor in an ovotestis of a 15-month-old heifer from which the reproductive tract was obtained from a local slaughterhouse. Histopathological evaluation revealed that the gonads were ovotestis, but also a coincidental sex cordstromal (granulosa cell) tumor. The sex chromosome type was determined to be XX, suggesting an XX ovotesticular disorder of sexual development with uterus and a female phenotype.

Key clinical message: Disorders of sexual development are common in mammals. Gonadal tumors in disorders of sexual development are rarely reported and have not been reported in cows.

CASE DESCRIPTION

The reproductive tract from a 15-month-old heifer of unknown breed was submitted by a veterinarian from a local slaughterhouse. The animal was identified through the traceability system as female and had the external genitalia of a female phenotype. The internal reproductive organs were abnormal in appearance. They were smaller than normal and had the appearance of testes. The organs were collected for pathology assessment due to a potential neoplasm.

Although the gonads had the appearance of testes, they were connected to a Y-shaped tubular structure resembling a uterus. On one side, the gonad measured 2.7 cm in diameter and contained a 2.2 × 1.8-centimeter mass with a firm and yellow appearance on the cut surface (Figure 1 A). Numerous coiled blood vessels were visible entering the gonad (Figure 1 A). The other gonad was 1 cm in diameter and irregular, and had similar coiled blood vessels. The tubular organs were smaller than normal (Figure 1 B). Histopathological and immunohistochemical analyses were completed to better assess the reproductive structures and the mass. Details of the immunohistochemical analysis are provided in Table 1 and in Table S1 (available online from: www.canadianveterinarians.net).

FIGURE 1.

Photographs of the abnormal reproductive structures in a 15-month-old cow. Arrows in (A) and (B) indicate the presence of the coiled blood vessels. A mass is noted at the extremity of one sample (arrowhead in A).

TABLE 1.

Results interpretation for immunohistochemical analysis.

Marker	Results	Used positive control	Expected results in sex cord tumors	Reference(s)
SOX9	Gonads ++ Tumor −	Bovine testis	+/−	Papanastasopoulos et al (25)
WT1	Gonads + Tumor +/−	Bovine testis	+/−	Zhao et al (26) Cathro et al (27)
Smooth muscle actin	Tumor −	Bovine skin	+/−	Horny et al (28) Hanson et al (29) Costa et al (30)
Pan-cytokeratin	Tumor + (30%)	Bovine skin	+/−	Anglickis et al (31) Gitsch et al (32) Hanson et al (29) Costa et al (30)
PLAP	Tumor −	Bovine placenta	−	Anglickis et al (31) Hanson et al (29)
S-100	Tumor −	Bovine skin	+/−	Anglickis et al (31) Hanson et al (29) Costa et al (30) Tanaka et al (33)
Vimentin	Tumor +++	Bovine skin	+	Horny et al (28) Anglickis et al (31) Hanson et al (29) Otis et al (34)

The gonads were composed of both male and female reproductive structures. Both gonads (right and left) had ovarian follicles with oocytes (primordial follicles) within an ovarian stroma (female) and large regions of hypoplastic seminiferous tubules ~100 μm in diameter (Figure 2 A, B). Immunohistochemical analysis proved positive for WT1 and SOX9, confirming these cells were sustentacular (Sertoli) cells (Figure 2 C, D). Both gonads had an immature epididymis (Figure 2 E). The tubular genitalia had the appearance of an immature uterus. The endometrium had scarce fibrous stroma and numerous small glands (Figure 2 F). Beside the uterus (on each side), there was a deferent duct with smooth muscle encircling a single layer of cuboidal epithelium (not shown).

FIGURE 2.

Histopathological and immunohistochemical evaluation of the abnormal reproductive structures. The presence of both female (A) and male (B) structures is noted in the gonads. Inset in (B) represents a high-magnification view (400×) of the tubular structures. The epithelium lining these tubules is both WT1- (C) and SOX9- (D) positive. Other structures include poorly differentiated epididymis (E) and uterus (F). Hematoxylin and eosin staining (A, B, E, F) or hematoxylin counterstaining (C, D). Scale bars = 100 μm (A, C, D) and 1000 μm (B, E, F).

The mass was surrounded by ovarian stroma. Follicles and seminiferous tubules were compressed at the periphery (Figure 3 A, B). The mass was well-circumscribed, expansile, and composed of numerous variably sized cystic structures with a mass of cuboidal, polygonal, or spindle cells (Figure 3 C). Occasionally, the cells surrounded a small globule of eosinophilic material resembling Call-Exner bodies (Figure 3 D, E). The cells of this mass had abundant and slightly eosinophilic cytoplasm. The nuclei were round and central with small amounts of chromatin. Anisocytosis and anisokaryosis were mild. There were 18 mitotic figures in 2.37 mm² (Figure 3 F). These cells were strongly positive for vimentin; about 30% of the cells were positive for WT1 and pan-cytokeratin but negative for SOX9, smooth muscle actin, PLAP, and S-100 (Figure 4).

FIGURE 3.

Histopathological analysis of the mass. The neoplasm (“T” in A, B) is localized within the ovotestis, compressing the normal structures (“G” in A, B). A dashed line (A, B) delineates the junction between the tumor and adjacent structures. The SOX9 immunohistochemistry shows positive cells (arrows in B), confirming the presence of the tumor within an ovotestis. The mass is composed of numerous follicular-like structures lined by multiple layers of round-to-polygonal cells (C). There are numerous structures compatible with Call-Exner bodies (arrows in D, asterisks in E). Frequent mitoses are noted (dashed arrows in F). Hematoxylin and eosin staining (A, C, D, E, F). Scale bars = 100 μm (B, D, E, F) and 1000 μm (A, C).

FIGURE 4.

Immunohistochemical analysis of the ovotestis mass. The cells are positive for vimentin (A), partially positive for pan-cytokeratin (B), negative for α-SMA (C), negative for S-100 (D), negative for PLAP (E), rarely positive for WT1 (arrows in F), and negative for SOX9 (G), counterstained with hematoxylin. Scale bars = 100 μm.

The sex chromosome was assessed using a newly developed qPCR assay based on previous reports (1,2). Briefly, paraffin-embedded tissues were deparaffined and DNA was subsequently extracted using a QIAamp DNA Mini kit (QIAGEN, Dusseldorf, Germany) following manufacturer recommendations. The qPCR assays were done using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems, Waltham, Massachusetts, USA) and conducted on a QuantStudio 3 apparatus (Applied Biosystems). The qPCR assays were optimized and validated using both fresh tissues and paraffin-embedded tissues from male and female bovine samples. The DNA input per reaction was of 50 ng, and cycling conditions were as follows: 50°C for 5 min, 95°C for 20 s, 40 cycles at 95°C for 3 s and 60°C for 30 s, and held at 4°C. The primers and probes designs used in the qPCR assays are listed in Table S2 (available online from: www.canadianveterinarians.net). The results indicated that all tissues tested in this case had an XX chromosome type. This disorder of sexual development (DSD) was therefore classified as XX sex chromosomes, bilateral ovotestes, immature uterus and epididymides, and deferent ducts. The mass was diagnosed as a sex cord-stromal tumor (SCST) and, based on phenotype and immunohistochemical analysis results, was subclassified as a granulosa cell tumor (GCT).

DISCUSSION

The cow described in this case report had both a DSD and a GCT. To the best of our knowledge, there are no previous published reports in the veterinary literature for this condition in cattle.

Granulosa cell tumors are the most common ovarian tumors in mares, cows, and cats. In cattle, they are diagnosed at any age but are most common in adults. Affected cows may be asymptomatic or exhibit nymphomania, male-like behaviors, or anestrus. In beef cattle, GCT is usually incidentally reported during meat inspection (3). Although GCT can secrete hormone, only few metastasize (4,5). Usually, antemortem diagnosis is based on a combination of clinical signs and additional tests with ultrasound and plasma biomarkers such as anti-Müllerian hormone (AMH), inhibin, and steroid hormones, and eventually confirmed with histopathological evaluation upon surgical excision or postmortem findings (6,7). The histomorphology of the tumor can vary greatly between cases, with patterns including solid, tubular, cystic, and micro- and macro-follicular subtypes (8). Some of these patterns can mimic other gonadal tumors, such as epithelial and germ cell tumors, and different immunohistochemical panels can be done for differentiation (Table 1). Sustentacular (Sertoli) cell tumor, another and an exceptionally rare ovarian SCST, is a differential diagnosis (9). Some GCT also exhibit Sertoli-like patterns (10,11). Both have similar immunohistochemical patterns. Therefore, the distinction between sustentacular (Sertoli) and GCT is challenging. In the case reported here, we used SOX9 and WT1 to confirm development of male structures in the ovotestes. Both are important transcription factors involved in male sex determination and are expressed early during testicular development (12). Although SOX9 should not be expressed in granulosa cells, WT1 is involved in granulosa cell development, and this may explain the expression pattern in this tumor (13). The diagnosis of sex cord-stromal (granulosa cell) tumor was made based on the overall histological appearance, morphology of the observed tumor, and different immunohistochemical results obtained, including the lack of SOX9 expression.

The classification of DSD currently used for animals follows the classification criteria for humans, and is based on sex chromosomes, presence or absence of SRY (male sex-determination gene) expression, gonadal type, and phenotypic combinations (14). The most common bovine DSD is a hematopoietic chimera (freemartin). Freemartinism affects females of twin pregnancies (estimated at ~2% of all pregnancies) with a male sibling. The female co-twin has an XX chromosomal complement and hematopoietic cells, and gonadal cells are XX/XY. Female reproductive structures are affected by male hormones when there is a placental vascular anastomosis. The male sibling is usually unaffected (5). The shared vasculature between the male and female siblings allows the male (XY) hematopoietic cells to survive and colonize the female hematopoietic organs. Therefore, both X and Y chromosomes can be identified in blood cells from female freemartins (1,15). In the case described here, the external genitalia were reported as female; however, we were unable to provide additional information regarding their morphological appearance. No blood was available for further analyses. Therefore, we could not completely exclude (or confirm) freemartinism.

There have been no reports of tumors in the ovotestes of cows. In other species, reports of the combination of DSD and gonad tumors exist but are scarce. A tumor developing in an ovotestis was reported in cats (16,17). In dogs, there have been occasional reports of gonadal tumors in combination with a DSD, and gonadoblastoma seems a recurring diagnosis (18,19). In one case, a testicular tumor was identified in the gonad of a female bearing a XX/XY leucocyte mosaicism (as observed in freemartinism) (20).

Determining risk factors for the development of neoplasia in ovotestes is difficult because similar cases are lacking. Abdominally retained gonads in males are more likely to develop SCST. In the present case, the neoplasm arose from the ovarian component. Ovaries are normally intra-abdominal and the prevalence of SCST in animals is very low. Different molecular mechanisms have been explored to better understand the pathogenesis of ovarian GCT. Among these, different signaling pathways involved in the regulation and effects of follicle-stimulating hormone (FSH), inhibin, activin, and AMH were studied (21). Abnormal development of the gonads and potential hormonal imbalance (such as for AMH) could therefore induce the development of GCT. However, evidence in human GCT suggest that AMH leads to activation of apoptosis, consistent with an antitumoral effect (22). The FSH is a potential candidate for the development of GCT due to its proliferative effects on granulosa cells (23). The concentration of FSH was higher in freemartin ewes, potentially due to the altered feedback response to the hypothalamo-pituitary axis because of reproductive-tract abnormalities (24). However, the lack of similar studies evaluating FSH secretion in cows with DSD prevents us from exploring this hypothesis.

In conclusion, it remains unknown why a neoplasm developed in the gonad of a cow with DSD. Thorough sex chromosomal, genetic, and morphological analyses are still required for a precise classification of DSD, and the diagnosis of SCST in an ovotestis is challenging. This study adds to the literature on gonadal tumors and DSD in cattle, as it provides a differential diagnosis for a mass in the gonad of an animal with a major DSD.

Funding Statement

G. St-Jean and C.A. Gagnon were financially supported by Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grants (nos. RGPIN2021-03021 and RGPIN-2017-05240, respectively). M.-J. Pesant was financially supported by a Fonds de Recherche du Québec-Nature et Technologies (FRQNT) scholarship (no. 324663). The Swine and Poultry Infectious Diseases Research Center is a research network financially supported by the Fonds de Recherche du Québec (FRQ).