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

Animal Science Papers and Reports vol. 28 (2010) no. 4, 381-388
Institute of Genetics and Animal Breeding, Jastrzębiec, Poland
SRY-negative, 64,XX sex reversal
in a Konik Polski horse: a case report
Kazimierz Jaszczak 1 *, Paweł Sysa 2 , Mariusz Sacharczuk 1 , Rafal Parada 1 ,
Katarzyna Romanowicz 3 , Magdalena Kawka 1 , Wiesław Jarmuż 1
1
Department of Molecular Cytogenetics, Polish Academy of Sciences
Institute of Genetics and Animal Breeding,
Jastrzębiec, 05-552 Wólka Kosowska, Poland
2
Faculty of Veterinary Medicine, Warsaw University of Life Sciences,
Nowoursynowska 159, 02-776 Warsaw, Poland
3
Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition,
Polish Academy of Sciences, 05-110 Jablonna, Instytucka 3, Poland
(Received March 18, 2010; accepted July 8, 2010)
A three-years old Konik Polski (Polish Primitive Horse) individual was diagnosed as having XX sex
reversal syndrome on the basis of female karyotype 64,XX, and the presence of two inter-abdominal
testes and ambiguous external genitals The individual exhibited aggressive, stallion-like behaviour.
The PCR analysis of the SRY gene revealed the absence of the Y chromosome sequences SRY in
DNA extracted from blood, gonads, skin and liver. This is the first reported case of an SRY-negative
XX- male sex reversal in a Konik Polski horse suggesting that it is sporadic case rather and not a
familial occurrence.
KEY WORDS: SRY-negative / sex reversal / Konik Polski horse
SRY-negative XX sex reversal is a disorder of gonadal development where
individuals having female 64,XX karyotype in the absence of the SRY gene (sex
determining region on the Y chromosome) develop testes or ovotestes. Such
individuals can be XX males or XX true hermaphrodites and are infertile. SRY-
*Corresponding author: k.jaszczak@ighz.pl
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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).
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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
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K. Jaszczak et al.
Photo 2. Metaphase plates of the XX sex reversal horse stained by CBG technique.
Photo 3. Electrophorogram showing the product of horse SRY fragment amplification (429 bp in length).
The absence of the SRY fragment was showed in the blood, testis, liver and skin of the Konik Polski
intersex. Taken together, the data are indicative of the absence of SRY gene in analysed individual
amounted from 1.9 to 3.2 ng/ml, respectively. The level of testosterone was higher
than in SRY-negative sex reversal of pony [Vaughan et al. 2000], but similar to that of
normal stallions [Kent et al. 1988].
Post mortem opening of the abdominal cavity revealed no evidence of uterine
tissue and ovaries, but two bilateral testes located near the internal inguinal ring.
The left testis measured 58 × 36 × 23 mm and the second 55 mm × 33 mm × 22
mm. Microscopic morphology of both gonads and epididymes was regular, however,
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Sex reversal in a horse
the size indicated their hypoplasia. Histological structure of both testes was similar.
Seminiferous tubules were built solely of supporting cells (Sertoli cells), with no sex
cells, even spermatogonia among them. In some lobules of gonads, Sertoli cells filled
up the entire cross-section area of seminiferous tubules, in other areas supporting cells
left considerable empty space of the tubule lumen. The tubules were encircled with
abundantly formed interstitial gland of testis (Leydig cells). In some regions it caused
the presence of far removed from each other single cross-sections of tubules (Photos
4 and 5). Gonads had the histological structure typical for the testes of cryptorchids.
In the epididymes the ducta epididymes were lined with pseudostratified columnar
epithelium forming various folds directed towards the lumen (Photo 6). Long
Photo 4. Left testis, seminiferous tubules encircled with overgrown interstitial gland (Leydig cells).
H and E; × 100.
Photo 5. Seminiferous tubules in the left testis of the intersex containing entirely supporting cells. No
spermatogenesis. H and E; × 250.
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K. Jaszczak et al.
Photo 6. Pseudostratified cylindrical epithelium with long stereocilia lining ductus epididymis.
H and E; × 40.
Photo 7. Cross-section of the right ductus epididymis with numerous epithelial folds.No sex cells in the
lumen of the duct. H and E; × 250.
stereocilia were seen on their upper surface. No sex cells were found in the lumen of
both epididymes (Photo 7). Thus the gonads were assessed as fully sterile.
The affected horse was diagnosed as having XX sex reversal syndrome on the
basis of female karotype 64,XX and presence of two inter-abdominal testes and
ambiguous external genitalia.
PCR amplification of the SRY gene was negative in all studied tissues, including
testes. The genetic mechanism by which testes development occurs in the absence
of the SRY gene is unclear. There is a possibility that other genes than SRY might be
responsible, but they have not been detected up to now [Buoen et al 2000, Vaughan et
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Sex reversal in a horse
al. 2001]. It is possible that mutations in an autosomal and X-linked genes allow the
testes development in the absence of SRY [Vaiman and Pailhoux 2000]. Some studies
have shown that Sox9 and Wnt-4 are candidates for sex reversal [Vainio et al. 1999,
Qin and Bishop 2005].
It is proposed that SRY-negative XX sex reversal syndrome is inherited in the
horse [Buoen et al. 2000]. The case described in this study is the first report on SRYnegative
XX sex reversal in the Konik Polski Horse. It is sporadic case rather and not
familial occurrence.
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