P. Parma

Classical and Molecular Cytogenetics of Disorders of Sex Development in Domestic Animals

The association of abnormal chromosome constitutions and disorders of sex development in domestic animals has been recorded since the beginnings of conventional cytogenetic analysis. Deviated karyotypes consisting of abnormal sex chromosome sets (e.g. aneuploidy) and/or the coexistence of cells with different sex chromosome constitutions (e.g. mosaicism or chimerism) in an individual seem to be the main causes of anomalies of sex determination and sex differentiation. Molecular cytogenetics and genetics have increased our understanding of these pathologies, where human and mouse models have provided a substantial amount of knowledge, leading to the discovery of a number of genes implicated in mammalian sex determination and differentiation. Additionally, other genes, which appeared to be involved in ovary differentiation, have been found by investigations in domestic species such as the goat. In this paper, we present an overview of the biology of mammalian sex development as a scientific background for better understanding the body of knowledge of the clinical cytogenetics of disorders of sex development in domestic animals. An attempt to summarize of what has been described in that particular subject of veterinary medicine for each of the main mammalian domestic species is presented here.

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.

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.

Characterization of a balanced reciprocal translocation, rcp(9;11)(q27;q11) in cattle

Cytogenetic analysis of a phenotypically normal young bull from Marchigiana breed revealed the presence of an abnormal karyotype. The observation of longer and smaller chromosomes than BTA1 and BTA29, respectively in all metaphases suggested the presence of a reciprocal translocation. RBG-banding confirmed this hypothesis revealing the involvement of BTA9 and BTA11. FISH analyses using cattle-specific BAC clones (474A12 and 293G09 for BTA9; 035D03 for BTA11) identified rcp(9;11)(q27;q11) in the two regions affected. Moreover analyses performed on both parents established the ‘de novo’ origin of the anomaly. Comparison with human homologue sequences (HSA6q24.3→q25.3 for BTA9q27 and HSA2q11.1→q12.1 for BTA11q11) revealed that both breakpoint regions are gene rich as up to date at least 200 genes have been localized in these regions. Thus, further analyses are required to identify the sequences disrupted by the breakpoints and to verify their consequences on rcp carrier phenotype.

Molecular mechanisms of sexual development.

Gonadal cellular organization is very similar in all vertebrates, though different processes can trigger bipotential gonads to develop into either testes or ovaries. While mammals and birds, apart from some exceptions, show genetic sex determination (GSD), other animals, like turtles and crocodiles, express temperature-dependent sex determination. In some groups of animals, GSD can also be overridden by hormone or temperature influences, indicating how fragile this system can be. This review aims to explain the fundamental molecular mechanisms involved in mammalian GSD, mainly referring to mouse as a major model. Conceivably, other mammals might show a molecular mechanism different from the commonly investigated murine species.

Clinical, genetic, and pathological features of male pseudohermaphroditism in dog

Male pseudohermaphroditism is a sex differentiation disorder in which the gonads are testes and the genital ducts are incompletely masculinized. An 8 years old dog with normal male karyotype was referred for examination of external genitalia abnormalities. Adjacent to the vulva subcutaneous undescended testes were observed. The histology of the gonads revealed a Leydig and Sertoli cell neoplasia. The contemporaneous presence of testicular tissue, vulva, male karyotype were compatible with a male pseudohermaphrodite (MPH) condition.

Reciprocal translocations in cattle: frequency estimation

Chromosomal anomalies, like Robertsonian and reciprocal translocations, represent a big problem in cattle breeding as their presence induces, in the carrier subjects, a well-documented fertility reduction. In cattle, reciprocal translocations (RCPs, a chromosome abnormality caused by an exchange of material between non-homologous chromosomes) are considered rare as to date only 19 reciprocal translocations have been described. In cattle, it is common knowledge that the Robertsonian translocations represent the most common cytogenetic anomalies, and this is probably due to the existence of the endemic 1;29 Robertsonian translocation. However, these considerations are based on data obtained using techniques that are unable to identify all reciprocal translocations, and thus, their frequency is clearly underestimated. The purpose of this work is to provide a first realistic estimate of the impact of RCPs in the cattle population studied, trying to eliminate the factors that have caused an underestimation of their frequency so far. We performed this work using a mathematical as well as a simulation approach and, as biological data, we considered the cytogenetic results obtained in the last 15 years. The results obtained show that only 16% of reciprocal translocations can be detected using simple Giemsa techniques, and consequently, they could be present in no <0.14% of cattle subjects, a frequency five times higher than that shown by de novo Robertsonian translocations. This data is useful to open a debate about the need to introduce a more efficient method to identify RCP in cattle.

Clinical, cytogenetic and molecular evaluation in a dog with bilateral cryptorchidism and hypospadias

The aim of this study was to estimate prognostic factors in a Dalmatian dog with bilateral cryptorchidism and hypospadias. Cytogenetic and molecular analyses revealed a normal karyotype (2n = 78,XY) and the presence of SRY, INSL3 and RXFP2 genes with a normal DNA sequence for SRY and RXFP2, while the INSL3 sequence differed slightly from the normal one due to a heterozygous nucleotide change involving amino acid 22 of the INSL3 dog precursor protein. Levels of plasmatic testosterone were only 0.01 ng/ml, while FSH and LH serum levels were not detectable. After the human chorionic gonadotropin (hCG) test, the serum testosterone level was 0.01 ng/ml. Therefore, the phenotypic aetiology of this subject can not be well-defined because cryptorchidism and hypospadias were frequent clinical features with high genetic heterogeneity.

Clinical, cytogenetic and molecular studies on sterile stallion and mare affected by XXY and sex reversal syndromes, respectively

Abstract Clinical, cytogenetic and molecular observations on a sterile stallion 2n = 65, XXY and a sterile mare 2n = 64, XY are reported. The XXY stallion was a pure case since all cells showed the same chromosome constitution. In the cells of mare XY, no SRY gene was found by both FISH- and molecular analyses. Both carriers show normal body conformation but were sterile because the stallion had no spermatozoa in the ejaculate, as revealed by microscope observation, and the mare showed the typical gonadal dysgenesis since both the uterus and ovaries were hypoplasic, as revealed by both rectal palpation and ultrasonic analysis. Although a mutation and / or deletion of SRY gene seems to be involved in the sex reversal, this syndrome is not yet fully understood. The possibility of other genes playing an important role in this syndrome and changes in the protein encoded by SRY are discussed.

FISH mapping in cattle (Bos taurus L.) is not yet out of fashion

Physical mapping of genes by fluorescence in situ hybridization (FISH) seems to be out of fashion in species whose assembled genome sequences are available. However, in this work we evidence the existence of errors in gene location in the Btau_4.0 assembly. We show thatDFNA5 andCHCHD6 genes are located on BTA4 and BTA22, respectively, instead of BTA10 and BTA3, as displayed by Btau_4.0. This report emphasizes the need to verify the data on physical localization of genes in the cattle genome (at least by taking into account comparative data reported in available papers) and the need to improve the cattle genome assembly. Our results indicate that FISH mapping in cattle is still useful.