Sry-negative XX Sex Reversal Research Articles

SRY-negative XX sex reversal in an English Cocker Spaniel: a case report

A SRY-negative XX male describes a female karyotype with testicular tissue resulting from abnormal gonadal development. In such cases male gonad formation takes place in the absence of a Y chromosome which supports the hypothesis that some other genes are also responsible for testicular development. The present case report describes a one-year-old dog brought to the hospital with a complaint of no oestrus cycle. Apparently normal masculine bitch had an enlarged clitoris with an os on radiograph. A gonado-hysterectomy was performed. Instead of ovarian structures both testicular masses were present and a normal complete bicornuate uterus with a normal cervix was observed. The histology of both gonads showed two testes, each with its ductus deferens and the uterus with typical histological structure. Cytogenetic evaluation of peripheral blood lymphocytes revealed a female with a normal karyotype (78, XX). Polymerase chain reaction (PCR) analysis of genomic DNA showed that the SRY gene was absent.

Sox9 duplications are a relevant cause of Sry-negative XX sex reversal dogs.

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.

SRY‐Negative XX Sex Reversal in a French Bulldog

Here, we describe a 3-month-old XX male French Bulldog. The diagnosis was based on the clinical signs, gonadal histology and cytogenetic analysis. Additionally, the dog was confirmed to be Sry negative by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR). Canine Sry-negative XX sex reversal is a disorder of gonadal development where individuals who have a female karyotype develop testes or ovotestes. To our knowledge, this case is the first XX male sex reversion described in a French Bulldog.

Polymorphisms in DMRT1 coding and promoter regions are probably not causative for swine sex reversal (XX,SRY-negative) syndrome

SRY-negative XX sex reversal is an inherited or sporadically occurring disorder, where testis development appears in the absence of the SRY gene. Although the molecular background of this intersexuality syndrome in pigs is unknown, it was proposed that familial cases might be inherited as a single autosomal recessive trait. Because DMRT1 (Doublesex and Mab-3 related transcription factor 1) is an autosomal locus in pig (SSC1q21), shows sexually dimorphic expression in swine gonads and has strong significance in vertebrate testis development, the molecular analysis of this gene was performed in previously reported three intersexes (38,XX, SRY-negative), the progeny of a single boar from a Polish farm. The first two exons encoding functional DM (double sex and mab-3) domain and the promoter region (the 5'flanking sequence) (altogether 3894 bp) were sequenced and compared with male and female control pigs (n = 16) and with publicly available sequences. Three different polymorphisms were found in the coding region, one Indel type polymorphism (DNA 142_144indelAGC) causing a deletion of an amino acid (protein S47_G48indelS) and two silent SNPs (DNA G432A and G492A). The promoter region seems to be highly polymorphic, since 17 SNPs and 5 indels were detected. However, the sequences of control males and females were concordant with those of the intersexes. These results indicate that DMRT1 is an unlikely candidate gene for SRY-negative XX sex reversal in pig.

Mutations in the RSPO1 Coding Region Are Not the Main Cause of Canine SRY-Negative XX Sex Reversal in Several Breeds

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.

Linkage to CFA29 Detected in a Genome-Wide Linkage Screen of a Canine Pedigree Segregating Sry-Negative XX Sex Reversal

Canine Sry-negative XX sex reversal is a disorder of gonadal development wherein individuals having a female karyotype develop testes or ovotestes. In this study, linkage mapping was undertaken in a pedigree derived from one proven carrier American cocker spaniel founder male and beagle females. All affected dogs in the analysis were XX true hermaphrodites and confirmed to be Sry negative by polymerase chain reaction. A genome-wide linkage screen conducted using 245 microsatellite markers revealed highest LOD score of 3.4 (marker CPH9) on CFA29. Fine mapping with additional microsatellites in the region containing CPH9 localized the Sry-negative XX sex reversal locus to a 5.4-Mb candidate region between markers CPH9 and FH3003 (LOD score 3.15). Insignificant LOD scores were found at genome-wide screen or fine mapping markers that were within 10 Mb of 45 potential candidate genes reported to have a role in mammalian sex determination or differentiation. Together, these results suggest that a novel locus on CFA29 may be responsible for sex reversal in this pedigree.

Genetics, genomics, and molecular biology of sex determination in small animals

The genomic revolution is beginning to facilitate advances in canine and feline medicine, as illustrated in our research. Our studies are focused upon identifying the gene mutation that causes canine Sry-negative XX sex reversal, a disorder of sex determination in which chromosomal females (78,XX) develop testicular tissue, becoming either XX true hermaphrodites with ovotestes, or XX males with bilateral testes. A genome-wide screen, using mapped markers in our pedigree of Sry-negative XX sex reversed dogs founded upon the American cocker spaniel, identified five chromosomal regions in which the causative gene may be located. The canine genome was used to identify the canine homologue of goat Pisrt1 and so determine that canine and caprine Sry-negative XX sex reversal are genetically heterogeneous. A second goal of our research is to determine the molecular mechanism by which the mutation causes testis induction. Thus far, we have reported gonadal Sry and Sox9 expression patterns in normal embryos, which have temporal and spatial patterns similar to those reported in humans, sheep, and pigs. Once gene mutations causing such inherited disorders are identified, DNA tests will become a part of general veterinary practice, advancing both diagnostic techniques and preventative medicine.

Exclusion of Candidate Genes for Canine SRY-Negative XX Sex Reversal

In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.

Exclusion of Lhx9 as a Candidate Gene for Sry-Negative XX Sex Reversal in the American Cocker Spaniel Model

XX sex reversal is known in 17 breeds of dogs. In the American cocker spaniel, it segregates as an autosomal recessive trait, and the affected animals lack the testis determining Sry gene. In the search for an autosomal gene that causes this trait, we considered the possibility of Lhx9, a gene encoding LIM homeobox containing transcription factor 9, as a candidate gene. An American cocker spaniel pedigree showing Sry-negative XX sex reversal phenotype was genotyped with an intronic Lhx9 microsatellite marker. Segregation of the Lhx9 marker in the pedigree indicated that a mutation in canine Lhx9 is not likely to be the cause of Sry-negative XX sex reversal. In addition, using the recently available 7.6X canine genomic sequence, we report the location and genomic organization of canine Lhx9.

Exclusion of DMRT1 as a candidate gene for canine SRY-negative XX sex reversal

DMRT1, which encodes a zinc finger-like DNA binding motif, is a well-conserved gene that is involved in testis differentiation in a variety of mammalian and non-mammalian vertebrates. The objective of this study was to determine whether a DMRT1 microsatellite marker allele is associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal generated from an American Cocker spaniel founder. Ten affected dogs and their parents and grandparents were genotyped. Four alleles at this locus and five different genotypes were found in this pedigree. All affected dogs inherited this trait from the foundation sire of this colony. Thus, the disease-causing mutation should be identical by descent in all affected dogs. Six affected dogs were found to have genotypes at this locus that were different from those of the founder sire. These results indicate that DMRT1 is an unlikely candidate gene for SRY-negative XX sex reversal in this model.

Sf1 andMis expression: Molecular milestones in the canine sex determination pathway

In mammals, the Y-linked Sry gene is normally responsible for testis induction. However, testes develop in the absence of Sry in human patients and animal models with Sry-negative XX sex reversal. The mechanism of testis induction in this disorder is presently unknown. Characterization of gene expression in normal embryos contributes to the framework within which the canine Sry-negative XX sex reversal model can be evaluated. The objective of this study was to add two molecular milestones to the canine sex determination pathway by determining the temporal and spatial expression patterns of Sf1 and Mis in normal urogenital ridges (UGR) at various gestational stages. The onset of Sf1 expression signifies the start of the sex determination period, whereas initial Mis expression identifies the end of the testis induction period. Sf1 expression in UGR was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and whole mount in situ hybridization (WMISH) at Carnegie stages (CS) 15 to 20. Canine sex determination begins at CS 15 with Sf1 expression in the emerging indifferent gonad. Gonadal Sf1 expression was detected in both sexes at all ages, and in the presumptive adrenal primordium at CS 15 and 17. At stages > or = CS 17, Sf1 expression was pronounced in male and female gonads. Mis expression, assayed by WMISH at CS 13.5-20, was observed only in male gonads > or = CS 18, indicating that the testis induction period ends at CS 18. The expression patterns of both genes are similar to those observed in humans and domestic animals.

Sry and Sox9 expression during canine gonadal sex determination assayed by quantitative reverse transcription-polymerase chain reaction.

Testis induction is associated with gonadal Sry and Sox9 expression in mammals, and with Sox9 expression in vertebrates where Sry is absent. In mammals, Sry might initiate testis induction by upregulating Sox9 expression; however, direct evidence supporting this hypothesis is lacking. Models of Sry-negative XX sex reversal (XXSR), in which testes develop in the absence of Sry, could provide the link between Sry and Sox9 in testis induction. To define the stages at which testis determination occurs in the canine model, Sry and Sox9 expression were measured in normal urogenital ridges (UGR) and gonads by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Testicular Sry expression rose continuously during canine developmental ages comparable to human carnegie stages (CS) 16-18, with maximal expression at CS 18. Sox9 was expressed in both male and female canine UGR up to CS 17, at which time testis expression became tenfold greater than in the ovary. Although Sox9 was detected by qRT-PCR in ovaries and mesonephroi of both sexes, expression was detected only in canine testes by whole mount in situ hybridization (WMISH). The timing of Sry and Sox9 expression is consistent with a role in testis determination: Sry expression begins at CS 16 in testes, followed by upregulation of Sox9 expression at CS 17. The quantity and temporal and spatial patterns of Sry and Sox9 expression in normal canine gonads are similar to those in humans, sheep, and pigs. These studies should provide the basis for understanding the mechanism of testis induction in the canine model of Sry-negative XXSR.

A 11.7-kb deletion triggers intersexuality and polledness in goats

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Sry-negative XX sex reversal in a pony: a case report

A three year old pony with sexually ambiguous external genitalia was found to have a normal female karyotype (64, XX) and bilateral inguinal testes. The PCR analysis of blood samples revealed the absence of the Y chromosome sequences SRY, eTSPY and ZFY. No Y chromosome sequences were identified in DNA extracted from the gonads. The mechanism whereby XX sex reversal occurs in the absence of SRY is unknown.

Sry-negative XX sex reversal in the German shorthaired pointer dog.

Present hypotheses indicate that a testis differentiation cascade in mammals is induced by Sry, a gene encoding a DNA binding protein of the high mobility group (HMG) class. In XX sex reversal, individuals lacking a Y chromosome develop testicular tissue. Sry translocation from the Y to the X chromosome has been found in some, but not all, of these individuals. XX sex reversal in the German shorthaired pointer dog may be a model of Sry-negative XX sex reversal in humans. The purposes of this study were to report the familial occurrence of sex reversal and determine whether the conserved Sry HMG box, the region of the Sry protein essential for testis induction, is present in genomic DNA of affected dogs. Canine Sry HMG box sequences were used as primers in polymerase chain reactions. A 104 bp Sry HMG box product was generated from normal males, but not from females or XX sex reversed dogs. Parallel control reactions using hypoxanthine phosphoribosyl transferase primers generated a 177 bp product from all dogs. The pedigree of affected dogs and the absence of Sry HMG box sequences in their genomic DNA suggest that this disorder is due to a mutant autosomal gene in the testis differentiation cascade.