SRY-negative, 64,XX sex reversal in a Konik Polski horse: a case ...

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K. Jaszczak et al. negative XX sex reversal syndrome was reported in numerous species of mammals [Vaiman and Pailhoux 2000]. The syndrome was diagnosed in at least 18 canine breeds [Meyers-Wallen 2006], in goats [Pailhoux et al. 2001a], and pigs [Pailhoux et al.1994, Pailhoux et al. 2001b,c]. Such a type of intersexuality was also recognized in several breeds of horses [Milliken et al. 1995, Meyers-Wallen et al. 1997, Bouen et al. 2000, Vaughan et al. 2001, Bannasch et al. 2007]. It was shown that SRY-negative XX sex reversal in mammals is caused by an autosomal recessive gene and may be an inherited disorder [Buoen et al. 2000]. This report describes one case of an SRYnegative XX sex reversal intersex phenotype in a Konik Polski horse with particular attention paid to the cytogenetic and molecular genetic aspects. Material and methods A three-year-old Konik Polski horse with sexually ambiguous external genitals and exhibited aggressive stallion-like behaviour was presented for histological, cytogenetic and molecular investigations. Konik Polski horses are a primitive native breed bred on the basis of a wild horse – tarpan. Clinically the horse had masculine appearance with the absence of scrotum and possesed ambiguous external genitals. Penis like clitoris was 7 cm long and protruded from the vulva lips (Photo 1). The Photo 1. External genitalia of a 64,XX reversal horse. urethral orifice was located in a female position and urination occurred through urethral fossa at the distal end of the penis. Transrectal ultrasonography to visualize the internal parts of reproductive organs and gonads was unrewarding, then this, and histological examination were conduced post mortem. Cytogenetic analyses were performed on the lymphocytes cultured in RPMI 1640 medium with pokweed mitogen. Chromosome preparations were analysed after C-banding [Sumner 1972]. The karyotype was determined based on examination of of 200 methaphase spreads (Photo 2). 382
Sex reversal in a horse For molecular genetic studies genomic DNA was isolated from whole blood of the intersex while a normal stallion and a normal mare were used as a control. Genomic DNA was also extracted from a section of the intersex testis, liver and skin with the DNeasy tissue kit (QIAGEN, Hilden, Germany). PCR primers were used to amplify a 429 bp SRY product: ESRYF002: 5’-CTTAAGCTTCTGCTATGTCCAGAGTATCC-3’ and ESRYR480: 5’-GCGGTTTGTCACTTTTCTGTGGCATCTT-3’; according to Hirota et al. [2001]. PCR was conducted using a PT-200 thermal cycler (MJ RESEARCH, USA) in a total volume of 25 μl that included 3μl 10 ng of genomic DNA, 20 μl injection quality water (POLPHARMA, Poland), 0.51μl dNTP (25 mM), 0.17 μl (25 μM) of each specific primer and 0.85 μl (2U/μl) Taq DNA polymerase (POLGEN, Poland) suspended in 2.62 μl of a buffer containing 100 mM Tris-HCl, 20 mM MgCl 2 and 500 mM KCl (POLGEN, Poland). The following conditions for the reaction were applied: 3.5 min at 94°C, followed by 32 amplification cycles (0:30 min at 94°C, 0:45 min at 62°C or 64°C, 1:30 min at 72°C) and the final elongation for 10 min at 72°C. The mixture was then cooled to 4 o C. PCR products were loaded onto a 1% agarose gel stained with ethidium bromide (APPLICHEM, Germany). Horizontal electrophoresis was carried out in the LKB-GNA 200 apparatus (PHARMACIA, Germany) at 100 mA/cm and 120 V for 1 h. An additional molecular-weight marker 1444-80 (pUC/Bsp R1/Taq) was added in the outside lane and gel running times were set so that fragments > 80 bp were retained on the gels. The presence of amplified SRY product was analysed visually in the transiluminator UV light (COLE-PARMER, France) and the software DNAProScan (USA). Blood samples for hormonal investigations were collected three times every two hours between 7.30 and 13.30 h. The concentration of serum testosterone was determined with a radioimmunoassay (RIA) method, as described by Stupnicki [1985]. The sensitivity of the assay was 0.6 ng/ml and the intra- and interassay coefficients of variations were 12.4 and 14.6%, respectively. Testes with epididymes were collected immediately post mortem. Organ specimens were fixed in Bouin fixative. After dehydration in a series of alcohols they were embedded in paraffin. Histological slides 5 μm thick, were stained with hematoxylin and eosin. Results and discussion The cytogenetic analysis of the intersex horse showed karyotype typical of a female equine (64,XX). In all cells examined (>200) no Y chromosome was observed (Photo 2). Absence of Y chromosome or part of it in several tissues was confirmed by PCR analysis. PCR amplification of the SRY gene was negative in the blood cells as well as in testicular-like tissue, liver and skin of the studied intersex horse (Photo 3). The concentration of serum testosterone in the sex-reversal horse, examined three times in blood plasma collected every two hours between 7.30 am and 01.30 pm 383
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SRY-negative, 64,XX sex reversal in a Konik Polski horse: a case ...

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