A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism.

Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid (n = 122) and control (n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3'UTR (16:23716202G>A) in KATA6, responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism (P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3'UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.

Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

The Case of Atypical Sexual Attractiveness in a Male Domestic Dog-A Case Study.

During the ovarian cycle in domestic dogs, females do not accept males during the first days of estrus but become attractive to males from the beginning of proestrus, with this attractiveness persisting until the end of the estrus phase. It is believed that increased estradiol is responsible for the female attractiveness to the males. In this paper we describe the case of strong, but atypical attractiveness of a castrated male to various, adult, intact males, influenced by the emitted semiochemical signals. Any significant changes in the level of hormones typically involved in the process connected with estrus and responsible for sexual arousal in the males were assessed. The case animal was a 4 year old castrated male Border Collie that was extremely attractive to various males, which presented high levels of sexual arousal, with intensive sniffing and licking of the preputial area, specific vocalization, increased salivation and, finally, mating attempts. Clinical examination of the castrated male revealed a lack of testes in the scrotum and abdominal cavity confirmed by USG. Laboratory tests indicated basal levels of estradiol, testosterone, and progesterone (15.23 pg/mL, <0.05 ng/mL, 0.25 ng/mL), and sex was confirmed via cytogenetic and molecular analysis. Chemical analysis (HS-SPME) of the urine indicated a huge similarity to the profile obtained from a bitch in estrus, with an elevated level of acetophenone, which has been previously postulated in the literature as being a characteristic of the estrus phase in female domestic dogs. This case presented very atypical sexual attractiveness, particularly when taking into account the basal levels of hormones which, according to current knowledge, are responsible for the creation of attractiveness. As a hypothesis requiring verification, we propose the idea of involvement of other hormones in the creation of incidental attractiveness or increased production of compounds responsible for attractiveness (sex pheromones) resulting from metabolic events unrelated to reproductive processes. To our knowledge it is the first described case presenting this phenomenon, which, with more detailed study, could shed new light on the process of creation of sexual attraction in the domestic dog.

Analysis of transcript and methylation levels of INSL3 and RXFP2 in undescended and descended dog testes suggested promising biomarkers associated with cryptorchidism.

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5'-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5'-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5'-regulatory regions of INSL3 and RXFP2, respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5'-flanking region of INSL3, and the ss7093349755 SNP in the 5'-flanking region of RXFP2.

Polymorphisms of MAMLD1, SRD5A2, and AR Candidate Genes in Seven Dogs (78,XY; SRY-Positive) Affected by Hypospadias or Cryptorchidism.

Knowledge of the molecular background of disorders of sex development (DSD) in dogs with normal sets of XY chromosomes (XY DSD) is very scarce. However, extensive studies have been carried out in humans, showing that polymorphisms and mutations of numerous genes, including SRY, MAMLD1, SRD5A2, and AR, are associated with or responsible for XY DSD. In this study, we analyzed the entire coding sequence of these genes in 7 dogs (78,XY) with ambiguous external genitalia (hypospadias, cryptorchidism, bifid scrotum, or rudimentary penis). The most common disorder was hypospadias (6 cases), followed by cryptorchidism (4 cases). The co-occurrence of both abnormalities was observed in 3 dogs. Polymorphisms were found in MAMLD1 (3 SNPs), SRD5A2 (5 SNPs), and AR (2 STRs and 1 SNP), while SRY was monomorphic. However, the distribution of the polymorphic variants in the DSD dogs and 11 control XY dogs did not differ significantly. Our study suggests that an association between the polymorphisms of the studied candidate genes and hypospadias or cryptorchidism is unlikely in dogs. We thus support the recent suggestion that hypospadias is not rare in this species, and moreover, we show that co-occurrence of hypospadias and cryptorchidism can be quite frequent.

Disorders of sex development in cats with different complements of sex chromosomes.

The genetic background of disorders of sex development (DSDs) in cats is poorly understood, due to a relatively low number of such studies in this species. Here we present three new DSD cases with different complements of sex chromosomes. The first, an Oriental Shorthair cat with a rudimentary penis, abdominal atrophic testicles and lack of uterus appeared to be a freemartin, since leucocyte chimerism XX/XY and a lack of Y-linked genes (SRY and ZFY) were observed in DNA isolated from hair follicles. XXY trisomy was identified in the second case, a tortoiseshell Devon Rex male cat with atrophic scrotal testicles and a normal penis. Finally, a European Shorthair cat with atrophic testicles in a bifid scrotum, rudimentary penis and a lack of uterus had XY complement, including Y chromosome of normal size and morphology. Also presence of eight Y-linked genes, detected by PCR, was confirmed. Due to the low testosterone level in this last patient, we searched for a causative mutation in two candidate genes (HSD3B2 and HSD17B3) involved in the metabolism of this steroid hormone. Altogether, five polymorphic sites in HSD3B2 and two in HSD17B3 were found, but none of them showed associations with DSD phenotype. We thus excluded a possibility that the causative mutation is present in these genes. In conclusion, we confirmed that analysis of the sex chromosome complement is a crucial step in diagnosis of DSDs. However, extensive molecular studies of the genes involved in sex development are needed to elucidate the causes of DSDs in cats with normal complements of sex chromosomes.

Hypospadias Is Not Rare in Dogs: Five New Cases, a Retrospective Study, and a Review of the Literature.

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.