Application of genomic and molecular methods to fundamental questions in canine and feline reproductive health.

Molecular tools are becoming increasingly available to investigate the genetic basis of reproductive disorders in dogs and cats. These were first successful in identifying the molecular basis of diseases inherited as simple Mendelian traits, and these are now being applied to those that are inherited as complex traits. In order to promote similar studies of reproductive disorders, we need to understand how we can play a proactive role in accumulating sufficient case material. We also need to understand these mutation discovery tools and identify collaborators who have experience with their use. The candidate gene and genomic approaches to mutation discovery in dogs are presented, including new sequencing methods and those used to confirm that a mutation has a role in disease pathology. As the final goal is to use our study results to prevent inherited disorders, we need to consider how we can promote efficiency in obtaining DNA test results and providing genetic counselling.

Sequence variations in equine candidate genes For XX and XY inherited disorders of sexual development.

Inherited disorders of sexual development (DSD) cause sterility and infertility in horses. Mutations causing such disorders have been identified in other mammals, but there is little information on the molecular causes in horses. While the equine genome sequence has made it possible to identify candidate genes, additional tools are needed to routinely screen them for causative mutations. In this study, we designed a screening panel of polymerase chain reaction primer pairs for 15 equine genes. These are the candidate genes for testicular or ovotesticular XX DSD and XY DSD, the latter of which includes gonadal dysgenesis, androgen insensitivity syndrome (AIS), persistent Mullerian duct syndrome and isolated cryptorchidism. Six horses with testicular or ovotesticular XX DSD and controls were screened. In addition, candidate genes for androgen insensitivity syndrome, persistent Mullerian duct syndrome and isolated cryptorchidism were screened in normal horses. While no sequence variants were uniquely associated with XX DSD, the 38 sequence variants identified can serve as intragenic markers in genome-wide association studies or linkage studies to hasten mutation identification in equine XX DSD and XY DSD.

A case of SRY-positive 38,XY true hermaphroditism (XY sex reversal) in a cat.

Investigation of abnormal sexual development in companion animals can allow for the elimination of inherited disorders from breeding populations while contributing to the understanding of the complex process of mammalian sexual development and differentiation. A 1-year-old mixed-breed cat, presented for neutering, was tentatively diagnosed as a male with bilateral cryptorchidism. During surgery, the surgeon identified gonads in an ovarian position and a complete bicornuate uterus. Both testicular and ovarian architecture in the gonads and Mullerian and Wolffian duct derivatives were identified histologically. The karyotype was that of a normal male (38,XY), and no causative mutation was identified in the feline SRY coding sequence amplified from genomic DNA. All features of the case were compatible with a diagnosis of SRY-positive 38,XY sex reversal, true hermaphrodite phenotype. To the authors' knowledge, this is the first report of this disorder in a domestic cat.

Review and update: genomic and molecular advances in sex determination and differentiation in small animals.

Inherited disorders of sexual development are important to identify as a cause of inherited infertility or sterility in humans and animals. Investigation of these disorders in dogs and cats can identify new mutations, allowing us to eliminate inherited disorders from breeding populations, while contributing to the understanding of mammalian sexual development and differentiation. This review updates an overview of normal mammalian sexual development while discussing disorders of sexual development at three consecutive levels, as errors in sex chromosome constitution, gonadal sex determination or phenotypic sexual development. The molecular mechanisms controlling sexual development and current molecular methods to identify causative mutations are illustrated in three specific examples of abnormal sexual development reported in small animals: XX sex reversal, Persistent Mullerian Duct Syndrome and cryptorchidism. Identification of causative mutations and development of practical tests to identify carrier and affected animals will provide effective mechanisms to reduce the prevalence of these disorders in small animals.

Unusual and abnormal canine estrous cycles.

Preovulatory serum progesterone concentrations are used to estimate the day of LH peak (day 0), not only to accurately time insemination and predict parturition, but to identify abnormal or unusual estrous cycles due to ovarian dysfunction. Early identification of these disorders is of therapeutic and economic importance. This review discusses anovulation, slow preovulatory progesterone rise, "split heat", insufficient luteal phase, and persistent estrus in the bitch. Some of these were temporary dysfunctions; with appropriate breeding management, pregnancy can be achieved. However, in other cases, these were signs of severe, permanent ovarian dysfunction associated with infertility, with potentially lethal sequelae.

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.

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.

Hematologic values in healthy neonatal, weanling, and juvenile kittens.

Blood samples were taken by jugular venipuncture from healthy kittens ranging in age from birth to 17 weeks. Complete blood cell counts, including RBC indices, provided data on reference blood values in kittens. Age-dependent RBC changes observed were decreases in PCV and hemoglobin (Hb) and mean cell Hb concentrations in neonatal kittens and a subsequent increase in PCV, Hb concentration, and RBC count in juvenile kittens. Leukocyte changes included an increasing WBC count from birth through the weanling period, increases in lymphocyte numbers in the neonatal and juvenile age groups, and increases in eosinophil numbers in the juvenile period.

XX true hermaphroditism in a dog.

XX True hermaphroditism was identified in a 5-month-old German Shorthaired Pointer with a large clitoris. The gonads were situated caudal to the kidneys at the cranial tips of the uterine horns, and were composed mainly of seminiferous tubules and interstitial cells and had ovarian follicles in the cortices. Each gonad had efferent tubules, a pampiniform plexus, fimbriae, and a uterine tube. The uterus was positioned normally in the abdomen and had no gross or histologic abnormalities. Giemsa-banded karyotypes revealed a normal female 78,XX chromosomal complement with no structural abnormalities.

Prostaglandin F2 alpha treatment of canine pyometra.

Immediate and long-term outcomes of prostaglandin F2 alpha treatment for canine pyometra were studied in 10 bitches. Examination of pretreatment uterine biopsy specimens, taken for histopathologic diagnosis and classification of disease severity, revealed either type III or IV pyometra. Dinoprost tromethamine (0.25 or 0.5 mg/kg of body weight, SC) was given once daily for 3 days. Bitches were bred at the first posttreatment estrus and monitored for a minimum of one year. When pure cultures of Escherichia coli (n = 3) or Staphylococcus aureus (n = 1) were obtained from the vagina, these bacteria also were found in the uterus. Pretreatment WBC counts often did not reflect the severity of histopathologic findings in the uterus, but posttreatment WBC counts were useful in monitoring response to treatment. Four bitches produced a litter within one year of treatment. Four bitches (40%) had recurrence of pyometra within one year of treatment, and these same bitches had another recurrence after an additional prostaglandin treatment. Three additional bitches had a recurrence by 27 months after therapy, establishing a total recurrence rate of 77% (7/9). Results suggested that subclinical disease may persist after treatment, with clinical recurrence during diestrus. Despite the high recurrence rate, it was concluded that this treatment is a practical treatment for canine pyometra when reproduction is desired.

Presumptive Sry-negative XX sex reversal in a llama with multiple congenital anomalies.

Multiple congenital abnormalities of the external genitalia consistent with XX sex reversal were detected in a juvenile llama. The llama had a typical female karyotype (74, XX) and did not have a Y chromosome, but a minute chromosome was detected. To determine whether a piece of Y chromosome containing the Sry gene might be located in a small translocation, DNA analysis by polymerase chain reaction was performed; the Sry gene was not detected. Histologic examination revealed ovarian tissue, whereas testicular tissue was not found. External genitalia were partially masculinized, indicating that the urogenital sinus, genital tubercle, and genital swellings had been exposed to androgens during development, although the dam had not received exogenous androgens. Testicular tissue in the ovaries may have been undetected or had regressed prior to birth, as has been reported in sex reversal in mice.

Testicular feminization in a cat.

Testicular feminization, caused by an inherited defect of the androgen receptor, was diagnosed in a domestic cat. Individuals affected with this syndrome are genetic males that have testes but fail to undergo masculinization because the internal and external genitalia cannot respond to androgens. The affected cat had the external appearance of a sexually normal female, but during surgery for ovariohysterectomy, only 2 abdominal gonads were found. Müllerian (uterus) or wolffian (epididymides) derivatives were not present. Only testicular tissue was found in histologic sections of the gonad. A normal male chromosome constitution (38,XY) was found in karyotypes prepared from lymphocyte cultures. High affinity binding of dihydrotestosterone was undetectable in fibroblasts cultured from genital skin of the affected cat, indicating that the cytosolic androgen receptor was nonfunctional. Pedigree analysis indicates that this is an X-linked disorder in cats, as it is in other mammals. Accurate diagnosis and genetic counseling are advocated to reduce the prevalence of the disorder.

XX sex reversal in a llama.

A 5-month-old llama was examined for evaluation of sexually ambiguous external genitalia. To determine the phenotypic, chromosomal, and gonadal sex of the llama, transrectal palpation and ultrasonography, contrast cystography, karyotype evaluation, laparoscopy, and necropsy were performed. A karyotype of 74,XX and finding of components of the müllerian duct system were suggestive of a female phenotype and chromosomal sex. On histologic evaluation, however, components of the wolffian duct system also were found, and the gonads were composed entirely of testicular tissue. The diagnosis was XX sex reversal, with a XX male phenotype.

Clinical approach to infertile male dogs with sperm in the ejaculate.

Categories of infertility are defined according to semen characteristics. Assessment of daily sperm output and carefully planned matings to determine the extent of infertility and to manage insemination to maximize fertility are described. The contributions of baseline laboratory data, evaluation of reproductive hormones, and testicular biopsy in determining the causes of infertility are discussed. Treatments of infertility secondary to systemic disease, infertility caused by accessory gland infections, and infertility associated with abnormal gonadotropin concentrations are reviewed.

Gonadal and sex differentiation abnormalities of dogs and cats.

The molecular steps in normal sexual development were largely discovered by studying patients and animal models with disorders of sexual development (DSD). Although several types of DSD have been reported in the cat and dog, which are often strikingly similar to human DSD, these have been infrequently utilized to contribute to our knowledge of mammalian sexual development. Canine and feline cases of DSD with sufficient evidence to be considered as potential models are summarized in this report. The consensus DSD terminology, and reference to previous terminology, is used to foster adoption of a common nomenclature that will facilitate communication and collaboration between veterinarians, physicians, and researchers. To efficiently utilize these unique resources as molecular tools continue to improve, it will be helpful to deposit samples from valuable cases into repositories where they are available to contribute to our understanding of sexual development, and thus improve human and animal health.

Trisomy-X with estrous cycle anomalies in two female dogs.

Two female dogs were presented with a history of abnormal estrous cycles and infertility, despite multiple breedings. Medical therapy to correct the cycle anomalies did not result in pregnancy. Cytogenetic analysis of blood lymphocyte cultures in each dog revealed three copies of the X chromosome in each cell, constituting a 79,XXX karyotype (trisomy-X). Both dogs were eventually ovariohysterectomised and histological evaluation revealed hypoplastic ovaries and an absence of normal follicular structures. However, partial or immature follicles were noted, which may have been sufficient to cause both females to initiate cycling. The history and clinical characteristics found in these dogs were compared to those described in three other dogs reported with trisomy-X, as well as those reported in other species. These findings highlighted the importance of cytogenetic studies in fertility evaluation and achieving a definitive diagnosis for infertility in the bitch.