Bilateral Ovotestes in a 78, XX SRY-Negative Beagle Dog

Introduction

Sexual determination in dogs takes place between days 36 and 46 of gestation.¹ At that stage, the embryo is sexually indifferent, a bipotential gonad arises from the urogenital ridge presenting both Wolffian and Müllerian ducts. In the absence of survival signals, the Wolffian ducts regresses and a female tract develops. Conversely, testicular secretions are essential for genital masculinization. Sertoli cells produce anti-Müllerian hormone (AMH), which is necessary for Müllerian duct regression and testicular descent through inhibition of the aromatase.² Leydig cells secrete insulin-like peptide-3, which is involved in the gubernaculum testis development. Testosterone is also produced that, either alone or after conversion to dihydrotestosterone (DHT), induces gubernaculum testis shortening, facilitating testis descent through the inguinal ring to the scrotum. Androgens stabilize the Wolffian duct system, and DHT masculinizes the external genitalia.¹

At the genetic level, in order to differentiate as a male, the embryo must potentiate the expression of the Y-linked sex-determining region Y (SRY) gene. That gene upregulates the autosomal gene SOX9, which induces the gonadal primordium to develop as testes. In the female, the absence of the SRY gene and the overexpression of negative regulators (such as DAX1 and R-SPONDIN 1 that inhibit SRY and SOX9, respectively) lead to ovarian development.^3,4

XX SRY-negative sex reversal is a disorder of gonadal development. Affected individuals have a female karyotype but their gonads may be entirely composed of testicular tissue. Those individuals are referred to as XX SRY-negative male pseudohermaphrodites. If the gonads contain both testicular and ovarian tissue they are referred to as XX SRY-negative true hermaphrodites. The true hermaphrodite can present with the testicular and ovarian tissue in different gonads or in the same gonad. The latter condition is referred to as ovotestis. There are three different forms of true hermaphroditism (1) bilateral, which presents bilateral ovotestes; (2) unilateral, which presents ovotestis on one side with contralateral testis or ovary; and (3) lateral true hermaphrodite, which presents with testis and a contralateral ovary. Those alterations have been reported in several canine breeds, including beagles.^5,6 However, those reports did not describe either the karyotype or the endocrine profiles of the affected individuals. That could have provided useful information about possible chromosomal alterations or sex hormone profiles, leading to a better understanding of the pathogenesis in beagles.^5,7

This report describes the findings on a case of bilateral ovotestes in a beagle. The authors included clinical signs, ultrasonographic and radiographic findings, an anatomopathological study, sex hormonal analysis, as well as conventional cytogenetic and polymerase chain reaction (PCR) analysis.

Case Report

A 6 mo old female beagle was presented for the evaluation of a finger-like structure protruding from the vulva. The owner reported that the dog had never been in estrus, often licked the vulva, and had difficulties urinating.

Clinical examination revealed a reddish structure protruding from the vulva consistent with an enlarged clitoris and vulvar inflammation. Vaginal cytology showed the presence of parabasal and small intermediate cells indicating anestrus and a relatively high number of neutrophils that suggested the presence of mild vaginitis.

A retrograde cystourethrovaginogram revealed a ratio of vestibulovaginal junction/vaginal height on the lateral vaginogram view of 0.12 (abnormal, <0.33) indicating a severe vestibulovaginal stenosis. Radiographic examination revealed the presence of an internal bone compatible with an os clitoris (Figure 1A). Ultrasound abdominal examination with a 7 MHz transducer revealed the presence of a tubular structure resembling a uterus that ran dorsal to the bladder and bilateral ovoid abdominal structures resembling gonads that were located caudal to the kidneys. The uterine structures had a diameter of 4 mm and showed a clear division of the uterine horns. The gonadal structures were different sizes (left, 14 mm × 9 mm; right, 9 mm × 5 mm) and had a homogenous and undifferentiated echogenic pattern.

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Cytogenetic analysis was performed as described by Selden et al. (1975).⁸ Briefly, 50 metaphase plates derived from cultured peripheral blood lymphocytes were analyzed after Giemsa staining. A female karyotype (78, XX) was observed on all plates.

A molecular analysis was performed to ascertain either the presence or absence of the SRY gene. Genomic DNA samples to be used as PCR templates for partial SRY amplification were prepared from normal fertile male and female dogs as well as the dog under investigation. The primers SRY-4F (5′-AAGGCCACGGCACAGAAAAAGTCAC-3′) and SRY-4R (5′-AAGAAGCGTCAGCGGACATCTGTG-3′) were used. The PCR reaction was performed after a denaturation step at 93°C for 3 min followed by 35 cycles of amplification (93°C for 30 sec, 56°C for 45 sec, and 72.2°C for 1 min).⁹ Finally, the products were separated by gel electrophoresis, stained with a fluorescent nucleic acid gel stain^a, and the presence of an SRY band was identified using a molecular weight marker. The results of the PCR amplification were negative in both the female control and the dog under investigation. A 271 base pair band corresponding to the SRY gene was observed in the male control dog (Figure 1B).

Serum steroid hormone concentrations (i.e., progesterone, testosterone, dihydrotestosterone, 17-β-estradiol) were evaluated^b. Paired samples were collected before and after gonadohysterectomy (GHE) using commercially available radioimmunoassay kits^c,d,e,f and standard procedures validated for the dog.^10,11 The hormone assays completed before GHE showed increased progesterone and dihydrotestosterone levels that returned to baseline 3 mo after surgery. The levels of testosterone that were within the reference ranges for an adult male before surgery decreased to basal levels postsurgically. The levels of 17-β-estradiol showed a small amount of variability but the values were always within the reference ranges for a male (Table 1).

TABLE 1 Sex Steroid Hormone Values Pre- and Postsurgically

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After GHE, the surgically removed tissue was routinely processed for histology and stained with hematoxylin and eosin (Figure 1C). The histology revealed that the gonads consisted of two different parts. One part resembled the testicle integrated by abundant connective tissue, and seminiferous-like tubules in which germ cells were not observed. The tubules appeared to be lined with cells with a large basal oval nucleus with cytoplasmic projections corresponding to Sertoli cells. In the interstitial space, round or polygonal interstitial cells (Leydig-like cells) were scattered between the seminiferous-like tubules (Figures 1D, E). The other part of the gonad displayed ovarian follicles (primordial, primary, and secondary) with oocytes and abundant ovarian interstitial cells (OICs) with round eosinophilic cytoplasm and round nuclei (resembling Leydig or theca cells) scattered between the follicles (Figure 1F). The outer surface of the gonad was covered with cuboidal cells, characteristic of ovarian surface epithelium. The uterine horns presented normal architecture and histological appearance with sparse glands. No epididymis or deferens ducts were observed. The diagnosis was 78, XX SRY-negative true hermaphroditism with bilateral ovotestes.

Discussion

True XX hermaphroditism is considered an uncommon congenital disorder defined by the presence of both testicular and ovarian tissue. Despite being reported in at least 15 canine breeds, many aspects of the etiology and the pathogenesis remain to be clarified.¹²

The cytogenetic analysis from this beagle revealed a normal 78, XX karyotype, which suggests that either chromosomal aneuploidy or mitotic nondisjunction on the Y chromosome could be ruled out as the etiologic agents of this case of XX SRY-negative true hermaphroditism. However, other possible causes, such as chimerism, could not be eliminated because that would have required multiple tissue biopsies from different areas of the body, which were not performed. A recent report by Switonski et al. (2011) described Robertsonian translocation in a XX SRY-negative Bernese mountain dog.¹³ Together, those observations support the generally accepted idea that this disorder may be genetically heterogeneous in dogs and could potentially present different genetic or chromosomal abnormalities for each breed.⁷

The genetic study revealed the absence of an SRY gene, which is in agreement with all hitherto tested 78, XX sex reversals, suggesting an SRY-independent mechanism for testicular tissue induction in dogs with an XX chromosome constitution.^5,12 It has been reported in other species that loss of function mutations in genes responsible for ovarian development (such as WNT4, FOXL2, and CTNNB1 mutations on the X chromosome that downregulate DAX1, RSPO1, Ad4BP/SF-1, and AMH) or mutations on an autosome chromosome that upregulate SOX9 can lead to partial testis determination and Sertoli cell differentiation without the SRY gene.⁴

As far as the study authors are aware, no literature exists concerning the steroid hormone profiles in patients with 78, XX SRY-negative bilateral ovotestes. The results from the patient described herein show that the sex steroid hormonal levels, except estradiol, decreased after GHE to basal levels, demonstrating that the XX SRY-negative genital tract has endocrine activity. The hormone assay completed before GHE revealed testosterone levels within the reference range for a male, whereas progesterone, estradiol, and DHT levels were high. Those results differ from data recently reported by Switonsky et al. (2011) who documented similar testosterone levels but lower progesterone and estradiol levels than were found in this study.¹³ However, this difference could be explained by the differences in the gonadal tissue observed in the patients because the case described by Switonsky et al. (2011) was a unilateral ovotestis that presented a testis.

In general, the hormone profile found in the patient described in this report correlated with the morphological findings but not with the SRY status. In this patient, the bilateral ovotestes could be an important source of progesterone and DHT. The high progesterone level can be explained by the presence of both testicular and ovarian tissue. In a true hermaphrodite, progesterone can be produced not only by Leydig cells, as an intermediate in the testosterone pathway, but also in the luteinized granulosa cells. The levels may vary both according to the amount of testicular and ovarian tissue and to the phase of the estrous cycle.¹⁴ The possibility that the endometrium could be an additional source of steroids remains to be investigated.

Despite normal testosterone levels, neither an epididymis nor vas deferens was found. That can be explained by the high amounts of progesterone and DHT, which are known to compete with testosterone for the receptor.¹⁵ On the other hand, the high DHT levels could be involved in the partial external masculinization observed. That was in agreement with the clitoromegaly and the presence of an os clitoris, commonly reported in the XX true hermaphrodite.¹² Another frequently reported abnormality, also present in this patient, was a normal morphologic and histological uterine structure. That was attributed to a lack of synchrony between the expression of AMH and its receptor during the development of the embryo.¹⁶ The sparse endometrial glands observed could be the result of the level of estradiol, which in this patient, was within the ranges for either an anestrous or prepubertal bitch.¹⁷

The hormonal profile of the patient is compatible with AMH production because one of biological effects of AMH is the biological inhibition of aromatase, which promotes the steroidogenic pathway towards an increase in androgens and progesterone.^2,18 In this patient, the presence of abundant OICs is another indicator that suggests the possibility of AMH production. Previous studies have shown that in the ovostestes, AMH can be produced not only by Sertoli cells but also granulosa cells. AMH could facilitate the formation of OICs as it is known to inhibit follicular development and OICs are thought to arise from the internal theca cells of atretic follicles.¹⁹

The abundance of OICs observed in this patient, a 6 mo old beagle, has also been described in a recent study of ovotestis in an 8 mo old cocker spaniel and a 3 yr old mixed-breed pit bull terrier, both XX SRY-negative true hermaphrodites¹⁴ The presence of abundant OICs in XX SRY-negative true hermaphrodite animals, regardless of their age or breed, may be associated with this alteration. Although the role of OICs remains to be studied, Groppetti et al. (2012) demonstrated P450c17 expression in OICs and have suggested their involvement in androgen synthesis. This is consistent with the high DHT levels detected before GHE in this patient. The role of the androgens as an etiologic agent remains to be clarified; however, it has been reported that exposure to an excess of testosterone in the gonads before sexual differentiation may be a possible cause of ovogenesis.²⁰ Further studies on endocrine profiles, aromatase activity, and the role of OICs can provide a new insight into hermaphroditism.

Conclusion

The dog presented an endocrine active bilateral ovotestes, that mainly produced progesterone and androgens, and a 2n = 78, XX chromosomal complement lacking the sex determining region Y gene, consistent with a diagnosis of true hermaphroditism.