Sex reversal syndrome in the horse: Four new cases of feminization in individuals carrying a 64,XY SRY negative chromosomal complement

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Contents lists available at ScienceDirect

Animal Reproduction Science

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ex reversal syndrome in the horse: Four new cases ofeminization in individuals carrying a 64,XY SRY negativehromosomal complement

abriel Anayaa, Miguel Moreno-Millánb, Monika Bugno-Poniewierskac,laudia Pawlinac, Alberto Membrilloa, Antonio Molinaa,ebastián Demyda-Peyrásb,∗,1

Laboratory of Animal Genomics, MERAGEM (AGR-158) Research Group, Department of Genetics, University of Córdoba, SpainLaboratory of Applied and Molecular Animal Cytogenetic, MERAGEM (AGR-158) Research Group, Department of Genetics, University ofordoba, SpainLaboratory of Genomics, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland

r t i c l e i n f o

rticle history:eceived 13 June 2014eceived in revised form9 September 2014ccepted 24 September 2014vailable online xxx

eywords:orse infertility diagnosishromosomal abnormalitiesicrosatellite analysis

n situ fluorescent hybridization

a b s t r a c t

Horses are characterized as having a greater rate of chromosomal abnormalities thanother species, which are mainly related to the sex chromosome pair and produce a seriesof different anomalies known as disorders in sexual development (DSD). In the presentstudy, three Pura Raza Espanola (PRE) and one Menorquín (MEN) horses were studiedand an incompatibility in their genetic and phenotypic sex were detected. Animals werekaryotyped by conventional and molecular cytogenetic analyses and characterized usinggenomic techniques. Although all individuals, were totally unrelated, these animals hadthe same abnormality (64,XY SRY negative DSD) despite having an anatomically normalexternal mare phenotype. Therefore, this syndrome could remain undiagnosed in a largepercentage of cases because the physiological and morphological symptoms are rare. In thepresent study, a slight gonadal dysgenesis was observed only in older individuals. Interest-ingly this chromosomal abnormality has been previously reported less than twenty times,

and never in the PRE or MEN horses. With the present research, it is demonstrated that theuse of genetic and cytogenetic diagnostic tools in veterinary practice could be an importantcomplementary test to determine the origin of unexplained reproductive failures amonghorses.

Please cite this article in press as: Anaya, G., et al., Sex

feminization in individuals carrying a 64,XY SRY negative chttp://dx.doi.org/10.1016/j.anireprosci.2014.09.020

∗ Corresponding author at: CN IV KM 396, Campus Rabanales CP 14071,regor Mendel Building, Cordoba, Spain. Tel.: +34 957 218509;

ax: +34 957 218707.E-mail addresses: [email protected], [email protected]

S. Demyda-Peyrás).1 Present address: Instituto de Genética Animal (IGEVET), University

f La Plata, Buenos Aires, Argentina.

http://dx.doi.org/10.1016/j.anireprosci.2014.09.020378-4320/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chromosomal aberrations are more often detected inhorses than in other domestic species and are normallyassociated with the sexual chromosome pair in morethan 95% of the cases (Iannuzzi et al., 2004; Lear andMcGee, 2012). Furthermore, a number of animals carry-

reversal syndrome in the horse: Four new cases ofhromosomal complement. Anim. Reprod. Sci. (2014),

ing these genetic diseases have no external phenotypicchanges (Demyda-Peyras et al., 2013). Due to this, a greaterpercentage of animals remains undiagnosed and the realprevalence of this abnormality in impacting important

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phenotypic traits is probably underestimated (Lear andBailey, 2008).

Sex reversal syndrome, which exists as an inconsistencybetween an individual’s genetic, gonadal and behavioralsex is the most common chromosomal pathology observedin veterinary practice (Villagómez et al., 2011). Currently,this genetic disease is classified according to the chro-mosomal complement detected in the individual (maleor female), which should be opposite to the phenotypicsex, and the presence/absence of sex determining region Y(SRY) gene (Mäkinen et al., 1999). According to Villagómezet al. (2011), there are four different possible conditions inhorses with disorders in sexual development (DSD): 64,XX;SRY positive males, which have not been reported to date,64,XX; SRY negative males and 64,XY mares either positiveor negative for the SRY. Among these, the 64,XY SRY nega-tive mares have been reported less than 20 times. Most ofthe cases were diagnosed in three recent studies performedin American (Raudsepp et al., 2010; Villagómez et al., 2011)and European horses (Bugno et al., 2003).

In the present study, four new cases of sex reversalin Pura Raza Espanola (PRE) and Menorquín (MEN) horsebreeds were analyzed for the first time with conventionaland molecular cytogenetic (fluorescent in situ hybridiza-tion – FISH) methods and genomic techniques, includingspecific microsatellite analysis to Equus caballus chromo-some X(ECAX)- and Y (ECAY)-linked markers as well as forthe SRY gene.

2. Materials and methods

2.1. Animals

Samples from four individuals phenotypically charac-terized as mares, were submitted for karyotyping to theapplied and molecular cytogenetic laboratory at the Uni-versity of Cordoba (Spain). Three of the animals (cases1, 2 and 3), were recorded in the PRE studbook and thefourth animal (case 4) was recorded in the MEN studbook.Analyses occurred between 2011 and 2013. Samples weresubmitted for karyotyping due to the abnormal resultsobserved in the mandatory parentage test (performed bythe breeders associations) in cases 1 and 2 or unexplainedinfertility in case numbers 3 and 4. The PRE breeders asso-ciation reported that there was no parentage relationshipamong the PRE horses (C1, C2 and C3) up to four genera-tions.

2.2. Physical examination and sampling procedure

All animals were examined by the official veterinary ser-vices of the respective breeder’s association. Cases 1 and2 (two fillies) were examined externally because of theyoung age of these individuals whereas rectal palpation andultrasonography were performed in the two older mares(cases 3 and 4). Blood samples were obtained by jugu-lar venipuncture using Tri-sodium ETDA BD VacutainersTM

Please cite this article in press as: Anaya, G., et al., Sex

feminization in individuals carrying a 64,XY SRY negative chttp://dx.doi.org/10.1016/j.anireprosci.2014.09.020

(MBL, Cordoba, Spain) for DNA isolation and sodium hep-arin BD VacutainersTM (MBL) for cell culture. Hair bulbs(n = 50) were aseptically collected for DNA isolation. At thetime of this study, C1 was euthanized by the breeder due

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to a personal decision, C2 was a 13-month old and C3 andC4 were 6- and 4-years of age, respectively.

2.3. Chromosome analysis

Lymphocyte cultures were established in RPMI1640medium for 72 h using standard procedures (Rodero-Serrano et al., 2013). Cells were incubated horizontallyin 12 mL tubes (Techno Plastic Products, Trasadingen,Switzerland), at 38 ◦C. After culture, cells were artificiallyarrested in the metaphase by incubation with colcemid(1 �g/mL, Sigma Aldrich) for 1 h; cells were then treated30 min with a hypotonic solution (25 min in 0.075 M KCl)and fixed twice in a cold methanol:acetic acid (3:1) mix-ture. The fixative was refreshed twice a day until the cellsuspension became transparent. The fixed cultures werestored at −18 ◦C until the time of use for evaluative pur-poses.

Chromosome spreads were obtained by applying 100 �lof the fixed solution onto a clean microscopic slide andair-dried at room temperature. Chromosome number wasdetermined in Giemsa stained slides using a Polyvar micro-scope (Reichert Jung, Austria) with 1250× magnificationin at least 150 analyzable metaphases (those with intactand non-overlapping chromosomes). Sex chromosomeswere identified and assessed using C-banding technique inat least 100 analyzable metaphases according to Sumner(1972).

2.4. In situ hybridization

Conventional chromosomal analysis was followed byin situ fluorescent hybridization with whole chromo-some painting probes (WCPPs) specific to ECAX andECAY. At least, 100 metaphases of each individual wereanalyzed. The probes were obtained by chromosomemicro-dissection followed by nonspecific PCR amplifi-cation with degenerated oligonucleotide primers (DOP)according to the routine laboratory protocol (Bugno et al.,2009). The probes were labeled either indirectly (ECAX)using biotin-16-deoxyuridine triphosphate (dUTP; RocheApplied Science, Penzberg, Germany) or directly (ECAY)using cyanine 3 dye (PerkinElmer, Waltham, USA). Denat-uration and hybridization of the probes and slides aswell as post-hybridization were conducted accordingto the standard protocol (Pienkowska-Schelling et al.,2006; Bugno et al., 2007). As for the indirectly labeledECAX probe, signals were detected and amplified usingavidin-fluorescein isothiocyanate (FITC) and anti-avidinantibodies (Sigma–Aldrich). Metaphases were stained withDAPI (Cambio, Cambridge, UK). Metaphase karyotypes ofeach individual (n = 100) were assessed in an Axiophot flu-orescence microscope (Carl Zeiss Microscopy GmbH, Jena,Germany) using Lucia software (Laboratory Imaging Ltd.,Prague, Czech Republic).

2.5. Molecular analyses

reversal syndrome in the horse: Four new cases ofhromosomal complement. Anim. Reprod. Sci. (2014),

Hair and blood were collected and the DNA from thesesamples was isolated with the QIAamp DNA mini kit fromall animals that were evaluated in this study and from a

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Fig. 1. Fluorescent in situ hybridization during the horse metaphase inanimals with a 64,XY karyotype. Horse metaphase spreads hybridizedusing two specific Equus caballus Y chromosome (ECAY) and Equus cabal-lus X chromosome (ECAX) fluorescent labeled probes according to Bugnoet al. (2009); 64,XY metaphase showing 62 blue autosomal chromosomes,

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are and a stallion for positive controls (Qiagen, Carls-ad, CA, USA). The ZFX/Y and SRY genes, two major sexelated genes located on the ECAY and ECAX, were assessedy polymerase chain reaction (PCR) according to Bugnot al. (2008) and Bannasch et al. (2007). The KIT gene wassed as positive control for the PCR reaction during the SRYmplification procedure (Haase et al., 2007). The amplifiedroducts were separated in 2% agarose gels stained withthidium bromide, by 70 min at 80 V and assessed underV light.

As a complementary study, eight microsatellite mark-rs (five located in the ECAX and three in the ECAY) wereenotyped in both, hair and blood DNA samples (Demyda-eyrás et al., 2014). Loci were amplified in two multiplexCR reactions and genotyped afterwards using an Appliediosystems 3130 xl DNA sequencer (SCAI genomics core,niversity of Cordoba, Spain). Allele sizes were determinedith the Genotyper 4.0 software package using LIZ 500 bp

nternal size standard (Applied Biosystems).

. Results

.1. Physical examination

All animals studied in this experiment showed a nor-al external morphology and a normally sized and shaped

ulva, clitoris and vaginal vestibule. However, C3 and C4howed an overall male body conformation. Reports of thewners and official veterinarians stated that the four ani-als did not demonstrate any female sexual behavior or

strous cycles. Cases 3 and 4 were examined internally,howing a morphologically normal uterus although slightlymaller compared to the normal dimensions of the respec-ive breed. The ultrasonographic assessment of the ovarieshowed homogenous structures with a diameter smallerhan expected. There were no signs of ovarian structuresuch as follicles or corpora lutea in either animal duringne (C4) or two (C3) reproductive seasons resulting in anal diagnosis of gonadal dysgenesis.

.2. Chromosomal assessment

The Giemsa-stained karyotype indicated there were4 chromosomes in the metaphase karyotypes that werenalyzed in the four animals. C-banding also showed theresence of one ECAX and one ECAY, compatible with aormal 64,XY male karyotype in all metaphase assess-ents that occurred. Results were confirmed by FISH. Alletaphase evaluations resulted in a small red ECAY signal

nd a large ECAX green signal being produced among theest of the chromosomes counterstained in blue (Fig. 1).ccordingly, the animals were diagnosed as 64,XY with aSD.

.3. Molecular characterization

Two different genes located in the sex chromosome pair

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feminization in individuals carrying a 64,XY SRY negative chttp://dx.doi.org/10.1016/j.anireprosci.2014.09.020

ere assessed. The ZFX/Y gene was amplified in all horses.ndividuals showing the 64,XY karyotype (the four casesnd the male control) showed a 604 bp specific ECAX bandnd a 553 bp specific ECAY band (Fig. 2). Only the ECAX

a large green signal from ECAX and a small red signal from ECAY. 1250×magnification. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

fragment was observed in the mare used as a female con-trol. The PCR amplification of the SRY gene was negative inthe four cases that were studied and in the female control,showing an 878 bp fragment from the KIT locus used as thecompetitive positive control (Fig. 3). Conversely, the malepositive control showed only a 242 bp band.

Results of microsatellite analysis are displayed inTable 1. All genotyped loci showed a single allele, indicat-ing the existence of only one ECAX and ECAY. Furthermore,results were the same in both, blood and hair DNA sam-ples, ruling out the presence of blood-cell chimerism. Thelack of variability observed in the ECAY linked loci wasremarkable being the same allele detected in each locus inall cases. Conversely, the variability observed in the ECAX-linked markers was much greater. These results confirm thepresence of one ECAY and one ECAX and the preliminarydiagnosis of a DSD.

4. Discussion

Chromosomal abnormalities are an important cause ofsubfertility or even sterility in horses. Among them, theDSD-linked pathologies are the most frequently detected(Villagómez et al., 2011). In the present study four newcases of 64,XY DSD were detected and characterized by adeletion surrounding the SRY region.

Previous reports have shown that animals with 64,XY

reversal syndrome in the horse: Four new cases ofhromosomal complement. Anim. Reprod. Sci. (2014),

DSD could be classified on the basis of morphology byexhibiting different phenotypes, from a female-like nor-mal appearance to individuals with male-like phenotypeand mixed gonadal tissue (Lear and McGee, 2012). This

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Table 1Analysis of a microsatellite panel linked to the sex chromosomes of the horse in three mares with DSD.

Locus Size range (bp) Case 1 Case 2 Case 3 Case 4 ECA

LEX003 194–214 214 206 198 198 XLEX026 300–314 306 314 312 306 XTKY38 105–131 129 109 129 105 XTKY270 154–172 168 154 168 168 XUCDEQ502 164–176 164 166 176 176 XEcaYA16 154 154 154 154 154 YEcaYH12 95 95 95 95 95 YEcaYM2 118 118 118 118 118 Y

Results obtained from a specific sex-related microsatellite panel (amplified according to Demyda-Peyrás et al., 2014) in the four cases. All the animals hada mono-allelic pattern in all the loci studied.bp: base pairs; ECA: Equus caballus chromosome.

Fig. 2. Results of the electrophoretic procedure for Zinc Finger X and Y geneamplification products in the individual animals from the present study.Two fragments (604 bp from ZFX and 553 bp from ZFY) were observedin the four studied animals (C1, C2, C3 and C4) and in the male posi-

Fig. 3. Results of the electrophoretic procedure of SRY and KIT gene ampli-fication products in the individual animals from the present study. A SRYpositive fragment (242 bp) was only observed in control male (Ma); a com-

tive control (Ma); only one fragment (ZFX, 604 bp) was observed in thefemale positive control (Fe); Wa: water negative PCR control; La: molec-ular weight marker.

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feminization in individuals carrying a 64,XY SRY negative chttp://dx.doi.org/10.1016/j.anireprosci.2014.09.020

classification appears to be genetically correlated with thepresence or absence of SRY, which acts as the basis fordetermining the type of expected anomaly. Whereas inindividuals with a normal SRY gene, testis tissue is usually

petitive 878 bp fragment (KIT) was observed in the four studied animals(C1, C2, C3 and C4) and in the male and female (Fe) positive controls; Wa:water negative PCR control; La: molecular weight marker.

present either as “testicle-like” structures or abdominaltesticles (Villagómez et al., 2011), in animals that are 64,XYSRY negative, DSD individuals are characterized by normalexternal female genitalia and, in most cases, gonadal dys-genesis (Lear and McGee, 2012). These animals are similarto horses that are 64,X0, in which the external gonads arenormal. Results from the present study are consistent withthese findings because all individuals had normal genitaliaand the mares with C3 and C4, the only ones which wereexamined internally, also had gonadal dysgenesis and lackof estrous behavior and reproductive cycles.

The SRY gene was identified as providing the differ-entiation signal of the male sex in animals, by initiatingthe differentiation of testis rather than ovary tissue devel-

reversal syndrome in the horse: Four new cases ofhromosomal complement. Anim. Reprod. Sci. (2014),

opment from early pluri-potential gonads (Sinclair et al.,1990). The activation of this gene upregulates a genecascade involving the autosomal SOX9 gene and the

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teroidogenic factor 1 (SF1), leading to the production ofnti-Müllerian hormone (AMH) by the testis and thus,reventing ovarian follicular development and the femalehenotype (De Santa Barbara et al., 1998). In humans, it

s well established that a simple mutation in SRY coulde responsible for sex reversal syndromes, among otherenetic diseases (Affara et al., 1993; Wagner et al., 1994).owever, most of the studies performed in horses show-

ng 64,XY DSD were conducted with a simple genomicechnique, i.e. PCR amplification of SRY (Abe et al., 1999;asegawa et al., 2000). Using this methodology, it is possi-le to determine only if the amplified product – in this casenly a portion of the gene – is absent or present. Due to usef the previously described technologies, the occurrence of

specific mutation that may alter the phenotype of an indi-idual, as was widely described in humans (Helszer et al.,013), cannot be determined.

Similarly, Ferguson-Smith (1965) demonstrated thatbnormal crossing-over and the abnormal recombinationf genes during embryo development could be responsibleor the loss of an SRY chromosomal section due to a mis-lignment produced in the post autosomal region (PAR), inhich the X and Y chromosomes are paired during humaneiosis. However, the detailed analysis of the horse chro-osome physical map has located the SRY in the ECAYq14,

ear to centromeric region (Raudsepp et al., 2004) and theAR on the terminal part of the long arm of the Y chro-osome, together with the AMELY and ZFY genes. Due to

hese previous findings, it is very unlikely that the samebnormality occurs in horses. However, recent studies havehown that recombination among sex chromosomes mayccur outside the pseudoautosomal regions (Rosser et al.,009), as demonstrated in felids (Slattery et al., 2000) ands suggested in horses (Raudsepp et al., 2010).

Most of the horses that have been diagnosed with chro-osomal abnormalities resulting in male-to-female sex

eversal were characterized as sporadic cases (Villagómezt al., 2011). However, in previous studies performed byent et al. (1986, 1988), some familiar inheritance patternsere described in Arabian sire lines. According to the PRE

nd MEN breeders associations, there were no reports oforphological or reproductive abnormalities among the

arental and maternal lines of the animals evaluated inhe present study and, as indicated previously, the animalsere completely unrelated. These results are consistentith more recent studies, in which the transmission of

4,XY SRY-negative DSD trough maternal or paternal linesas not demonstrated (Bugno et al., 2003; Villagómez et al.,

011). It could be possible that the lack of molecular stud-es during the time period when many previous studiesccurred when the inheritance of the 64,XY SRY negativeSD syndrome was proposed, could have led to a misinter-retation of results. For example, a recent study by Révayt al. (2012) found that there is a hereditary component inome cases where the 64,XY SRY-positive DSD is detected.t the time of the initial studies (middle eighties), thereere no diagnostic methods to differentiate SRY positive

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feminization in individuals carrying a 64,XY SRY negative chttp://dx.doi.org/10.1016/j.anireprosci.2014.09.020

nd negative animals (Pailhoux et al., 1995), and therefore,his could lead to a misinterpretation of the results.

There are few studies regarding 64,XY SRY negative DSDndividuals including a molecular determination of SRY

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gene (Abe et al., 1999; Mäkinen et al., 2001; Bugno et al.,2003; Villagómez et al., 2011) However, to our knowledge,a comprehensive genomic analysis in these individualswas previously performed in only 13 horses by Raudseppet al. (2010). It is noteworthy that 11 of these animalshad the same ECAY deletion surrounding the SRY gene. Itwas observed that certain genetic similarities existed inthe present and previous studies. However, following thepreviously used methodology, the exact extension of thedeleted sequence could not be determined in the presentstudy. In agreement with the previous study, an identicalrepeated sequence was detected in the present study thatwas located on both sides of the SRY gene. For this reason, itis not possible to determine which side of the deletion wasamplified in the PCR reaction (3′, 5′ or both) and therefore,the exact extension of the sequence could not be deter-mined. While it is true that there is still no valid explanationfor this phenomenon, it is suggested that some of a spe-cific processes could lead to similar deletions at the samegenome and in several individuals, without any particulargenetic relationship being necessary for these deletions tooccur.

5. Conclusion

The 64,XY SRY negative DSD genetic disorder could bea prevalent genetic abnormality among horses. However,it could remain undiagnosed mainly because animals withthis genetic disease are anatomically normal and exhibitsymptoms of unexplained infertility or small gonads.Therefore, the use of molecular tools to diagnose thissyndrome in a proper, rapid and efficient way could sub-stantially improve the number of horses diagnosed, witheconomic and time efficiencies for veterinarians and breed-ers.

Conflict of interest

The authors do not have any conflict of interest.

Acknowledgements

We thank the Asociación Nacional de Criadores deCaballo Espanol that generously provided the samplesand veterinary reports of the animals. We also thank toLic. Adriana Di Maggio by the language editing. SebastiánDemyda Peyras is supported by a Cesar Milstein grant(639/14; Ministerio de Ciencia, Técnica e Innovación Pro-ductiva, Argentina). The present study was supported bythe Laboratory of Molecular and Applied Animal Cytoge-netic, MERAGEM (AGR-158) Research Group, Universityof Cordoba, Spain and by the Laboratory of Genomics,National Research Institute of Animal Production, Poland.

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