Supplementary Material for: XX SRY-Negative True Hermaphrodism in Two Dogs: Clinical, Morphological, Genetic and Cytogenetic Studies

This work aimed at giving a deeper insight into peculiar cases of intersexuality occurring in dogs and known as XX true hermaphrodism due to the existence of both testicular and ovarian tissue in one or both gonads in the presence of an XX chromosome constitution. Clinical, histological and genetic approaches were used in the study of an 8-month-old Cocker Spaniel dog and a 3-year-old mixed-breed Pitbull, both showing a female phenotype, clitoromegaly and male behavior. A normal female karyotype (2n = 78,XX) was noticed, and polymerase chain reaction failed to detect SRY in genomic DNA obtained from peripheral blood lymphocytes of both dogs. The reproductive tract was removed by standard ovariohysterectomy and processed for histology. Thereafter, a normal female phenotype was reconstructed by vaginoplasty. Histological examination revealed bilateral ovotestis in both cases: the gonads showed immature testicular parenchyma containing seminiferous tubules, Sertoli and Leydig cells, but no signs of spermatogenesis, together with differently developed ovarian follicles containing oocytes. In the ovotestes, steroidogenesis was detected by P450c17-immunoreactivity in Leydig cells as well as in theca cells, whereas no MIS-immunoreactivity was shown by the Sertoli cells. Genital tracts of Wolffian and Müllerian origin co-existed in both subjects. Both dogs belong to the very rare cases in which testicular tissue develops in the absence of the key gene, SRY. Up to date very few genetic events have been associated with this abnormal sexual differentiation: SOX9 over-expression and RSPO1 mutation. Nevertheless, neither of them has been found in these dogs.本研究旨在深入剖析犬类中发生的特殊间性病例，这类病例因在XX染色体组型背景下，单个或双侧性腺同时存在睾丸组织与卵巢组织，故而被称为XX真两性畸形（XX true hermaphrodism）。本研究针对两只表现为雌性表型、阴蒂肥大且兼具雄性行为的犬只展开：一只为8月龄可卡犬，另一只为3岁混种比特犬，并采用临床、组织学与遗传学方法对其进行分析。检测结果显示，两只犬的核型均为正常雌性核型（2n=78,XX）；从其外周血淋巴细胞中提取的基因组DNA经聚合酶链式反应（Polymerase Chain Reaction, PCR）检测，未检出性别决定区Y（Sex-determining Region Y, SRY）基因。研究人员通过标准卵巢子宫切除术摘除两只犬的生殖道，并进行组织学处理；随后通过阴道成形术重建其正常雌性表型。组织学检查结果显示，两只犬均存在双侧卵睾体：其性腺可见未成熟的睾丸实质，内含生精小管、支持细胞与间质细胞，但无精子发生迹象；同时可见不同发育阶段的卵泡，内含卵母细胞。在卵睾体中，通过P450c17免疫反应性检测发现，间质细胞与卵泡膜细胞均存在类固醇生成活性，而支持细胞未显示出抗苗勒管物质（Müllerian Inhibiting Substance, MIS）免疫反应性。两只犬的生殖道均同时存在午菲管（Wolffian duct）与苗勒管（Müllerian duct）来源的结构。本研究涉及的两只犬均属于极为罕见的病例：在缺失关键基因SRY的情况下，睾丸组织仍得以发育。截至目前，仅有两类遗传事件被报道与该异常性别分化相关：SOX9过表达与RSPO1基因突变，但本研究的两只犬均未检出这两类异常。