Original Research
Diagnosis of XX/XY Blood Cell Chimerism at a Low Percentage in Horses

https://doi.org/10.1016/j.jevs.2018.06.016Get rights and content

Highlights

  • Disorders of sexual development (DSDs) are still a main cause of economic loss in horse breeding.

  • Genetic and cytogenetic analyses on various tissues are essential in horse DSDs.

  • DNA profiling on blood alone is not enough to detect all cases of cell chimerism.

Abstract

Disorders of sexual development (DSDs) are common in horses and cause economic loss in horse breeding. Thus, it is important to develop methods for unambiguous and fast identification of affected horses shortly after birth, as well as those that may propagate the condition to the next generation. Genetic causes of DSDs are multivarious and still little known, and thus development of diagnostic tests requires accumulating knowledge about individual cases and their etiologies. In particular, it is necessary to perform clinical, ultrasound, surgical, histological, cytogenetic, and genetic analyses with close attention in all the affected individuals. This report describes the case of a XX/XY chimeric horse with reproductive apparatus abnormalities and a very low percentage of XY cell in blood highlighting that to avoid undiagnosed case of cell chimeras, above all when studying DSD cases, it is essential to perform both genetic and cytogenetic analyses possibly on more than one tissue.

Introduction

Reproduction and fertility are important concerns in horse breeding, and early identification of horses with congenital conditions that may lead to reproductive problems will bring a big benefit to horse industry.

Although cytogenetic and molecular tools have been developed for this purpose, most horses carrying disorders of sexual development (DSDs) are identified when they have already grown up, causing economic loss to the breeders, and in most cases, molecular causes remain unknown [1], [2], [3], [4]. This is because of the limited knowledge about the molecular mechanisms regulating early development and sexual differentiation.

DSDs are among the main causes of horse subfertility or sterility. A variety of phenotypes are associated with this condition ranging from a phenotypically normal mare with gonadal dysgenesis to a horse with ambiguous external genitalia and internal male and female organs [5]. In horses, four types of DSDs have been diagnosed up to now: (1) sex chromosome abnormalities (63,X; 64,XX/64,XY; 65,XXX; 65,XXY; etc.,); (2) 64,XX SRY-negative with DSD; (3) 64,XY SRY-positive with DSD; (4) 64,XY SRY-negative. In horses, XX SRY-positive DSD has never been reported, probably because the SRY gene is located far from the pseudoautosomal region; thus, it is less susceptible to meiotic errors between the sex chromosomes compared to, for example, humans where SRY translocation to the X chromosome can occasionally occur [6].

XX/XY chimerism is classified as a chromosome abnormality, and it has been diagnosed in the main livestock species and in humans. It is caused either by the exchange of hematopoietic stem cells through placental circulation between dizygotic twins (blood chimerism) or by the fusion of two zygotes or embryos into a single individual at the very early stages of development (true chimerism) [7], [8]. Phenotypic and physiological effects due to this condition are very variable and depend on both the causes and the affected species.

XX/XY chimerism has been rarely diagnosed in horses; this is either because twin pregnancy (the main origin of chimeras) causes serious economic loss as a result of a high rate of abortion and a tendency for poor postnatal development in the few foals that survive to term; thus, it is an unwanted condition normally terminated once detected [9], [10]. However, large-scale DNA profiling or cytogenetic survey of horse populations [11], [12] suggests that the available clinical data underestimate the actual prevalence of these cases.

From a scientific point of view, a procedure able to detect chimeras rapidly and early and to differentiate those caused by placental vascular anastomosis in a twin pregnancy rather than an early fusion of two zygotes or embryos would be very useful. In fact, the different phenotypes due to chimerism, and mainly those XX/XY, are a useful starting point for understanding the mechanism of sexual differentiation in mammals, but for this purpose, it is necessary to correctly identify affected animals as early as possible in their lifetime so that the development of the reproductive apparatus can be followed during all the growth phases allowing to accumulate new knowledge. Moreover, it is necessary to establish the cause of the chimerism; in twin pregnancy with placental anastomosis between the twins, one of them may miscarry without breeder's knowledge.

Vascular connections between placentas of heterosexual twins cause in ruminants the so-called free-martin syndrome [7], [13] in which the female twin is sterile due to malformations of the reproductive apparatus, while in equine blood, chimeric heterosexual twins are both phenotypically and physiologically healthy and fertile [9], [14]. This difference is probably due to the fact that placental vascular connections responsible for free-martin syndrome in ruminants and other species occur after the sexual differentiation of the equine [14].

A different condition is found when chimerism is due to the fusion of two zygotes or embryos. In this last case, the phenotype may be normal or ambiguous genitalia may be observed [15].

This report describes the diagnosis of the first case of a 64,XX/64,XY chimeric horse, showing a reproductive apparatus in which only male reproductive structures have been developed, with the aim to highlight the need of both cytogenetic and genetic analyses in all animals in which a correct genetic evaluation is required (clinical and DSDs cases, breeders).

Section snippets

Case

A 15-month-old Italian Saddlebred horse, registered as filly, was submitted to clinical evaluations due to abnormal conformation of external genitalia (Fig. 1) and stallion-like behavior. On physical examination, the horse showed a small penis of 11 cm in length in the ventral perineal region without scrotum and an underdeveloped mammary gland (Fig. 1). Urination occurred through a urethral fossa at the distal end of the penis. Transrectal ultrasonography did not allow to visualize internal

Histopathologic Analyses

Both of the testes were composed of low number of small and hypocellular seminiferous tubules that lacked germ cells and spermatozoa and were lined by Sertoli cells, often with frothy, vacuolated apical cytoplasm (Fig. 2A). Sertoli cells extended from the undulating basement membrane and protruded into the lumen. The interstitial tissue, separating the tubules, was apparently increased because of the reduced number of tubules and was composed by well-developed fibrovascular stroma with embedded

Discussion

Reproductive apparatus abnormalities observed in a 15-month-old horse led to deepen the clinical case by performing clinical, ultrasound, surgical, histological, cytogenetic, and genetic analyses with close attention.

Anatomical and histopathological findings of this horse indicate that during embryo development, the pathway of formation of the male genital apparatus has been correctly activated. This has led to testes formation and to their migration in inguinal canals. However, the genital

Conclusion

SRY PCR positivity with 64,XX normal karyotype found in a 15-month-old horse with abnormalities of reproductive apparatus led to deepen Giemsa-stained (without banding) karyotyping and C-banding test allowing to diagnose XX/XY chimerism, and subsequently, microsatellite genotyping on DNA from gonadal tissue allowed to classify the case as a tetragametic chimera. To date, there are very few reports of XX/XY horse chimeras [11], [22], [23] with malformed genital apparatus, and this is the first

Acknowledgments

The authors wish to thank Dr. Borriello G. and Dr. Riccardi M.G. (Istituto Zooprofilattico Sperimentale del Mezzogiorno) and Prof. Chiofalo V. and Dr. Cosenza M. (UnireLab s.r.l.) for their technical support.

References (23)

  • G. Anaya et al.

    Sex chromosomal abnormalities associated with equine infertility. validation of a simple molecular screening tool in the PurebredSpanish Horse

    Anim Genet

    (2017)
  • Cited by (0)

    Animal welfare/ethical statement: No experimental animals have been used for this work.

    Conflict of interest statement: None of the authors has any conflict of interest to declare.

    1

    These authors contributed equally to this work.

    View full text