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Disorders of sex development (DSD) are a well-known cause of sterility and reduced fertility in dogs. Here, we report on 6 new cases of DSD in French bulldogs diagnosed by us within the last year. The animals were phenotypical females who underwent detailed diagnosis due to the presence of an enlarged clitoris or a penis-like structure with atypically located urethral orifice (hypospadias). A female karyotype (78,XX) and lack of Y-linked genes (SRY and ZFY) were observed in 5 cases, which were classified as XX (SRY-negative) DSD. Gonads histology could be performed in 4 XX DSD cases: testicular tissue was observed in 3 cases and ovotesticular tissue in 1 case. In the last case, leukocyte chimerism (XX/XY) was detected alongside an ovotestis with epididymis and fallopian tubes. This case was classified as freemartinism. Our study and a literature review showed that heritable XX (SRY-negative) DSD is quite common in French bulldogs, but spontaneously developed freemartinism can be also diagnosed. In conclusion, we postulate that the description of the sexual phenotype of DSD females (enlarged clitoris and presence of testicular tissue) does not distinguish between hereditary XX DSD and nonhereditary freemartinism. Cytogenetic and molecular analyses are thus necessary to correctly diagnose such cases. This is especially important for the French bulldog breed, as a rapid increase in its population could spread hereditary DSD.

Hypospadias, the abnormal position of the urethral orifice, is considered a rare congenital malformation of the reproductive organs in male dogs. We present 5 new cases of hypospadias - 2 of the penile type in German Shepherd Dogs and 3 perineal types in a Bavarian Mountain Hound, a French Bulldog, and an American Staffordshire Terrier. Other abnormalities (rudimentary or underdeveloped penis, incompletely formed preputial sheath, and bilateral cryptorchidism) were also observed. Molecular analysis of all cases revealed the presence of Y-linked genes (SRY and ZFY). Cytogenetic and histological analysis could be performed for only 2 cases: a normal male sex chromosome complement (78,XY) and spermatogenetically inactive testicles were observed. A retrospective search for hypospadias in 19,950 medical records of male dogs from a single veterinary clinic in Poland (2006-2017) was also performed. Altogether, 10 reports of penile hypospadias were found (0.05%). The majority of the reports concerned German Shepherd Dogs (8 cases among 1,511 male dogs of this breed), and thus, the estimated incidence of hypospadias in this breed was 0.5%. Moreover, we performed a review of 26 cases of canine hypospadias reported in the years 2004-2017. Our study and the review of the literature suggest that hypospadias is not rare in dogs and that some breeds (such as German Shepherd Dog and Boston Terrier) may be prone to this disorder.

Testicular or ovotesticular disorder of sex development (DSD) in genetic females (78,XX; SRY-negative) has been reported quite frequently in numerous dog breeds and is usually diagnosed due to the presence of female external genitalia with an enlarged clitoris. The molecular background of this disorder, diagnosed also in human and other mammals, is not fully understood. However, it has recently been proposed that a copy number variation (CNV) in the region upstream of the SOX9 gene is associated with it. We described a rare case of this disorder in a French Bulldog with abdominal testes and male external genitalia (a slightly malformed penis). FISH studies showed a female karyotype, lack of a translocation involving the Y chromosome, and a distinct size variation in the CNV region (CNVR) upstream of the SOX9 gene, located on chromosome 9 (CFA9). A large FISH variant on a single CFA9 and a lack of the variant on its homologue was observed. Surprisingly, in the mother of this DSD dog, 2 normal-sized variants were identified which means that the CNV in the DSD dog was de novo. Our observations are in agreement with earlier suggestions that a high number of copies at the CNVR upstream of SOX9 may be associated with this type of DSD.

A disorder of sexual development (DSD) of dogs with a female karyotype, missing SRY gene, and presence of testicles or ovotestes is quite commonly diagnosed. It is suggested that this disorder is caused by an autosomal recessive mutation; however, other models of inheritance have not been definitely ruled out. In an earlier study it was hypothesized that the mutation may reside in a pericentromeric region of canine chromosome 23 (CFA23). Three positional candidate genes (CLASP2, UBP1, and FBXL2) were selected in silico in the search for polymorphisms in 7 testicular or ovotesticular XX DSD dogs, 8 XX DSD dogs of unknown cause (SRY-negative, with enlarged clitoris and unknown histology of gonads), and 29 normal female dogs as a control group. Among the 15 molecularly studied dogs with enlarged clitoris there were 3 new cases of testicular or ovotesticular XX DSD and 4 new cases of XX DSD with unknown cause (histology of the gonads unknown). Altogether, 11 (including 10 novel) polymorphisms in 5′- and 3′-flanking regions of the studied genes were found. The distribution analysis of these polymorphisms showed no association with the DSD phenotypes. Thus, it was concluded that the presence of the causative mutation for testicular or ovotesticular XX DSD in the pericentromeric region of CFA23 is unlikely.

Hypospadias is rarely reported in dogs. In this study we pre-sent 2 novel cases of this disorder of sexual development and, in addition, a case of hereditary sex reversal in a female with an enlarged clitoris. The first case was a male Moscow watchdog with a normal karyotype (78,XY) and the presence of the SRY gene. In this dog, perineal hypospadias, bilateral inguinal cryptorchidism and testes were observed. The second case, representing the Cocker spaniel breed, had a small penis with a hypospadic orifice of the urethra, bilateral cryptorchidism, testis and a rudimentary gonad inside an ovarian bursa, a normal female karyotype (78,XX) and a lack of the SRY gene. This animal was classified as a compound sex reversal (78,XX, SRY-negative) with the hypospadias syndrome. The third case was a Cocker spaniel female with an enlarged clitoris and internally located ovotestes. Cytogenetic and molecular analyses revealed a normal female karyotype (78,XX) and a lack of the SRY gene, while histology of the gonads showed an ovotesticular structure. This case was classified as a typical hereditary sex reversal syndrome (78,XX, SRY-negative). Molecular studies were focused on coding sequences of the SRY gene (case 1) and 2 candidates for monogenic hypospadias, namely MAMLD1 (mastermind-like domain containing 1) and SRD5A2 (steroid-5-alpha-reductase, alpha polypeptide 2). Sequencing of the entire SRY gene, including 5′- and 3′-flanking regions, did not reveal any mutation. The entire coding sequence of MAMLD1 and SRD5A2 was analyzed in all the intersexes, as well as in 4 phenotypically normal control dogs (3 females and 1 male). In MAMLD1 2 SNPs, including 1 missense substitution in exon 1 (c.128A>G, Asp43Ser), were identified, whereas in SRD5A2 7 polymorphisms, including 1 missense SNP (c.358G>A, Ala120Thr), were found. None of the identified polymorphisms cosegregated with the intersexual phenotype, thus, we cannot confirm that hypospadias may be associated with polymorphism in the coding sequence of the studied genes.

A Bernese mountain dog was subjected for clinical evaluation due to the presence of ambiguous external genitalia (enlarged clitoris). Anatomical and histological studies revealed the presence of one testicle, one ovotestis and a uterus. This dog was classified as a female-to-male sex reversal, with 2 normal X chromosomes and a lack of the Y chromosome-linked genes SRY and ZFY. It is the first case of this syndrome in this breed. Apparently a Robertsonian translocation, rob(5;23), was also identified in this dog and it is again the first case of this type of chromosome abnormality in this breed, as well as the first case of co-occurrence of the sex reversal syndrome along with a centric fusion in the dog. Since on the canine chromosome 23 (CFA23) 3 genes (FOXL2,PISRT1 and CTNNB1) involved in the sex determination process are present, further cytogenetic FISH studies were carried out with the use of BAC probes specific for this chromosome. It was found that a pericentromeric fragment of CFA23 was deleted as a result of the centric fusion. We hypothesize that a cis regulatory sequence for the sex determination genes on CFA23 (e.g. proximally located CTNNB1) is present in the deleted fragment. Thus, a causative mutation responsible for this sex reversal syndrome may reside on CFA23.