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.     

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.     

Sry-negative XX sex reversal in the American cocker spaniel dog

The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley-Liss, Inc.     

Sry‐negative XX sex reversal in purebred dogs

The gene responsible for testis induction in normal male mammals is the Y‐linked Sry. However, there is increasing evidence that other genes may have testis‐determining properties. In XX sex reversal (XXSR), testis tissue develops in the absence of the Y chromosome. Previous polymerase chain reaction (PCR) assays indicated that autosomal recessive XXSR in the American cocker spaniel is Sry‐negative. In this study, genomic DNA from the breeding colony of American cocker spaniels and from privately owned purebred dogs were tested by PCR using canine primers for the Sry HMG box and by Southern blots probed with the complete canine Sry coding sequence. Sry was not detected by either method in genomic DNA of affected American cocker spaniels or in the majority (20/21) of affected privately owned purebred dogs. These results confirm that the autosomal recessive form of XXSR in the American cocker spaniel is Sry‐negative. In combination with previous studies, this indicates that Sry‐negative XXSR occurs in at least 15 dog breeds. The canine disorder may be genetically heterogeneous, potentially with a different mutation in each breed, and may provide several models for human Sry‐negative XXSR. A comparative approach to sex determination should be informative in defining the genetic and cellular mechanisms that are common to all mammals. Mol. Reprod. Dev. 53:266–273, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

XX sex reversal in the American cocker spaniel dog: phenotypic expression and inheritance

Summary This study was conducted to define the range of phenotypic expression and mode of inheritance of XX sex reversal in the cocker spaniel dog. Breeding experiments produced F1, F1BC, and F2 generations in which 29 XX true hermaphrodites and 3 XX males were defined by chromosome constitution, serial histologic sections of the gonads, and examination of the internal and external genitalia. In XX true hermaphrodites, the most common combination of gonads was bilateral ovotestes, followed by ovotestis and ovary, then ovotestis and testis. The amount of testicular tissue in the two gonads was closely correlated within each true hermaphrodite. The distribution of testicular tissue within ovotestes of true hermaphrodites was consistent with the hypothesis that testicular differentiation is initiated in the center of the gonad and spreads outward. XX males had bilateral aspermatogenic testes and the internal ducts and external genitalia were more masculinized than in true hermaphrodites. Results of breeding experiments are consistent with autosomal recessive inheritance, the affected phenotype being expressed only in dogs with an XX chromosome constitution. The phenotypic expression and mode of inheritance of this disorder is compared to XX sex reversal in humans and other animals.     

Sry-negative XX true hermaphrodite in a Basset hound

A true hermaphrodite was diagnosed in a 7-mo.-old Basset hound. The diagnosis was based on the clinical signs, the histology of the gonads and the karyogram. Additionally, the dog was tested for the Y-linked gene Sry, which was negative. The Basset hound presented here is compared to other XX sex reversed animals described in the literature. In man, XX sex reversal is a heterogenous condition. The pathogenesis in Sry-negative individuals is not understood. Thus Sry-negative animals could serve as an animal model of the human disease.     

Generalized progressive retinal atrophy of Sloughi dogs is due to an 8-bp insertion in exon 21 of the PDE6B gene

We investigated the gene encoding the beta subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.     

Recessive sex-determining genes in human XX male syndrome

Maleness is normally inherited as a dominant trait (a single copy of the Y chromosome induces testicular differentiation of the embryonic gonad), but our genealogic study of three XX males in one pedigree indicated an autosomal recessive mode of male inheritance. Subsequent study revealed the presence of H-Y antigens in the three XX males and in their mothers, and suggested that excess H-Y may be found in the fathers. Inasmuch as H-Y loci have been mapped to the human Y chromosome, these data favor the view that H-Y structural loci comprise a family of testis-determining genes, and that Y autosome (or Y-X) translocation can generate either dominant or recessive modes of XX sex reversal, depending upon the particular portion of H-Y genes transferred.     

Familial (9;11)(p22;p15.5)pat translocation and XX sex reversal in a phenotypic boy with cryptorchidism and delayed development

We describe a patient with the co-occurrence of a familial 9;11 reciprocal translocation and an XX sex reversal. The patient had cryptorchidism, delayed development, dysmorphic features and attention deficiency hyperactive disorder (ADHD). The proband's karyotype was 46,XX,t(9;11)(p22;p15.5) and he was positive for SRY gene. The father was found to be the carrier of the similar translocation. The co-occurrence of XX sex reversal and autosomal reciprocal translocation has not been described previously. The possible reasons for the manifestation of features other than those found in XX sex reversal is described.     

Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1 , a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.     

Inherited XX sex reversal in the cocker spaniel dog

Summary Nine XX true hermaphrodites and two XX males were discovered in a family of American cocker spaniels. The true hermaphrodites were partially-masculinized females with ovotestes; the XX males had malformed male external genitalia and cryptorchid aspermatogenic testes. Wolffian and Mullerian duct derivatives were present in both true hermaphrodites and XX males. All four sires of sex-reversed dogs were normal XY males; five of the dams were anatomically normal females and one was an XX true hermaphrodite. A second true hermaphrodite reproduced as a female, producing anatomically normal offspring.All matings that produced sex-reversed offspring were consanguineous. Matings of the parents of sex-reversed cocker spaniels to normal beagles with no family history of intersexuality produced only normal offspring. Examination of G-banded karyotypes of the affected animals, their parents, and siblings, revealed no structural anomalies of the chromosomes that were consistently associated with sex-reversal.In assays for serologically-detectable H-Y antigen, the group of XX true hermaphrodites and the group of XX males had mean levels of the antigen not significantly different from that in normal male controls. Female parents of sex-reversed dogs and some of their female siblings were typed H-Y antigen positive, but the mean level of the antigen in this group was less than that of normal male controls.It is proposed that XX sex reversal in cocker spaniels is due to a mutant gene which when homozygous in females, results in a level of H-Y antigen similar to that found in normal males and the gonads develop as ovotestes or testes. When the gene is heterozygous in females, the level of serologically-detectable H-Y antigen is lowr than that found in normal males and the gonads develop as normal ovaries. The persistence of Mullerian structures in the presence of testicular tissue suggests that Mullerian inhibiting substance is deficient or ineffective in its action in this condition.Supported by NIH Postdoctoral Fellowship IF32 HL05515, University of Pennsylvania Genetics Center Grant, No. GM 20138, and NIH grants AI-19456, HD 17049, and HD 14357; and a grant from the Mrs. Cheever Porter Foundation.     

Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes

The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession number AY623815.     

Increase of cortisol levels after temperature stress activates dmrt1a causing female‐to‐male sex reversal and reduced germ cell number in medaka

In vertebrates, there is accumulating evidence that environmental factors as triggers for sex determination and genetic sex determination are not two opposing alternatives but that a continuum of mechanisms bridge those extremes. One prominent example is the model fish species Oryzias latipes which has a stable XX/XY genetic sex determination system, but still responds to environmental cues, where high temperatures lead to female‐to‐male sex reversal. However, the mechanisms behind are still unknown. We show that high temperatures increase primordial germ cells (PGC) numbers before they reach the genital ridge, which, in turn, regulates the germ cell proliferation. Complete ablation of PGCs led to XX males with germ cell less testis, whereas experimentally increased PGC numbers did not reverse XY genotypes to female. For the underlying molecular mechanism, we provide support for the explanation that activation of the dmrt1a gene by cortisol during early development of XX embryos enables this autosomal gene to take over the role of the male determining Y‐chromosomal dmrt1bY.