Clinical, cytogenetic and molecular studies on sterile stallion and mare affected by XXY and sex reversal syndromes,

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1 CARYOLOGIA Vol. 57, no. 4: , 2004 Clinical, cytogenetic and molecular studies on sterile stallion and mare affected by XXY and sex reversal syndromes, respectively Iannuzzi L. 1 *, G.P. Di Meo 1,A.Perucatti 1,M.Spadetta 2,D.Incarnato 1,P.Parma 3,A. Iannuzzi 2,F.Ciotola 2,V.Peretti 2,G.Perrotta 4 and R. Di Palo 2 1 National Research Council (CNR), Laboratory of Animal Cytogenetics and Gene Mapping, Naples, Italy; 2 Department of Animal Production and Food Inspection, University Federico II, Naples, Italy; 3 Applied Epigenetic Laboratory, Experimental Institute Lazzaro Spallanzani, Lodi, Italy; 4 Blood Group Laboratory (LGS), Soc. Coop. A.R.L., Cremona, Italy Key words: cytogenetics, horse, sterility, sex reversal, XXY. Abstract Clinical, cytogenetic and molecular observations on a sterile stallion 2n=65, XXY and a sterile mare 2n=64, XY are reported. The XXY stallion was a pure case since all cells showed the same chromosome constitution. In the cells of mare XY, no SRY gene was found by both FISH- and molecular analyses. Both carriers show normal body conformation but were sterile because the stallion had no spermatozoa in the ejaculate, as revealed by microscope observation, and the mare showed the typical gonadal dysgenesis since both the uterus and ovaries were hypoplasic, as revealed by both rectal palpation and ultrasonic analysis. Although a mutation and/or deletion of SRY gene seems to be involved in the sex reversal, this syndrome is not yet fully understood. The possibility of other genes playing an important role in this syndrome and changes in the protein encoded by SRY are discussed. INTRODUCTION While autosomal numerical aberrations are easily visible in domestic animals due to their deleterious effects on body conformation, sex chromosome abnormalities are more tolerated by species since carriers show generally normal body conformation and external genitalia. This is due to the presence of a single copy gene in the sex chromosomes and inactivation of genes present in one of two X chromosomes. However, numerical sex chromosome abnormalities are often correlated with sterility or hypofertility, especially in female carriers. Reproductive problems related to sex chromosomal abnormalities are more common in horse than in other domestic species. Indeed, about 94.5% of chromosomal abnormalities found in horse involve sex chromosomes, 93% of which involve only sex chromosomes and 1.5% involve the sex chromosomes together with autosomes (Chowdhary and Raudsepp 2000). * Corresponding author: CNR-ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Via Argine 1085, 80147, Naples, Italy; phone ; fax ; L.Iannuzzi@iabbam.na.cnr.it Among sex chromosomes, while reversal syndrome, known as Swyer s syndrome in humans, is relatively common in horses, especially in XY mares, (Kent et al. 1986, 1996; Power 1986; Pailhoux et al. 1995; Thrommershausen-Bowling et al. 1987; Scherer et al. 1998; Makinen et al. 1999, 2001; Bugno et al. 2003), the XXY condition, known as Klinefelter syndrome in humans, in very rare in this species, especially the pure XXY. Indeed, only two pure cases have been reported in this species (Kubien et al. 1993; Makinen et al. 2000). In the present study clinical, cytogenetic and molecular observations on a sterile stallion 2n=65, XXY and a sterile mare 2n=64, XY are reported. In our knowledge, this is the third case of pure XXY found in the horse and the first two horse clinical cases studied by both clinical and cytogenetic approaches in Italy. MATERIALS AND METHODS Case 1. A three-year-old trotter stallion of the Bardigiano breed raised in Liguria (northern-italy) was the third offspring of a 12-year-old mare and a 5-year-old sire. Since four mares were mated because they did not conceive, the breeder required clinical

2 clinical cytogenetics in horse 401 and cytogenetic analyses on the stallion. Semen sample was collected once when stallion covered a mare. The stallion was later mated and it was not possible to obtain further analyses. Case 2. A 6-years-old mare from a Trotter breed raised in province of Caserta (southern Italy) was seemingly normal, with normal clitoris and vulva, but it never conceived. Hence clinical, cytogenetic and molecular studies were performed. Ultrasonic analyses on the mare were performed with a Sonovet 600 ultrasonic scanner, equipped with a MHz 7.5 linear probe. Cell cultures and chromosome banding techniques - One ml of peripheral blood sample was cultured in 8 ml of RPMI 1640 medium (Gibco-BRL), 1 ml of fetal cal serum (Gibco-BRL), 0.1 ml of penicillinstreptomycin (Gibco-BRL) and 0.2 ml of pokeweed mitogen (Sigma). Cultures were incubated for about 72 h at 37.7 C. Two types of cell cultured were performed: culture A, without the addition of base analogue; culture B with the addition of both 15 µg/ml of Bromodeoxyuridine (BrdU, Sigma) and Hoechst (30 µg/ml, Sigma) during the last 6.5 hours of culture as follows. After about 48 h, thymidine (300 µg/ml, Sigma) was added to cell cultures for 17 hours for cell cycle block at the S-phase. Cell block was removed by washing cells twice with Puck s saline solution and recovering cells for 6.5 h in fresh medium containing BrdU and Hoechst as reported above. Slides from normal cultures were treated for CBA-banding (Iannuzzi, 2003), while slides from R- banded chromosome preparations were treated for RBA- and RBG-banding (Iannuzzi, 2003). For each animal, 100 and 30 metaphases were analysed for C- and R-banding, respectively. Some slides from case 2 (sex reversal) were also treated for FISH-technique by using a bovine BACclone containing the SRY-gene (Iannuzzi et al., 2001b). Karyotypes were arranged according to the Iscnh (1997). DNA extraction and PCR amplification - Genomic DNA was extracted from peripheral blood of fertile male and female horses, as well as from a XY mare by using a standard commercial kit (Quiagen blood kit, Valence, CA). The PCR amplifications, performed in a PTC-100 thermocycler (MJ Research, Reno, NV), were conducted in a 30 µl-volume containing 1.5 mm MgCl 2, 200 µm of each dntp, 1 µl of each primer (at concentration of 5 µm) and 2 units of AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). Primer sequences (SRYfrw: 5 -TACCACCCTCCTCTTCAACG-3 and SRYrew: 5 - CGAAAATAGAGCCGAACAGG-3 ) were designed on the sequence described by Hasegawa et al. (1999, GenBank accession number AB004572). The positive PCR amplification results in a 795 bp PCR product. RESULTS Case 1. The stallion was taller than other stallions of the same breed and age weighing 350 Kg. Size of penis, scrotum and testes appeared a little smaller compared to the size of the animal which showed normal sex libido. The volume of ejaculate was 38 ml and the colour was brownish. No spermatozoa were found when analysing the ejaculate under the microscope. All 100 cells treated for CBA-banding which we examined were 2n=65, XXY (Figure 1). Hence, this stallion was affected by pure XXY syndrome, as confirmed by sequential RBG/CBA-banding (not shown). The stallion was retained to be sterile and, for this reason, mated. Case 2. The mare showed normal stature and weight (Kg 450) when compared with mares of the same age and breed. External genitalia were normal (Figure 2A). Both uterus and ovaries were hypoplasic, as revealed by both rectal palpation and ultrasonic analyses (not shown). In particular, the right ovary was mm 11x16 and the left was mm 9x13. No other structures, such as follicles or corpus luteum bodies, were detected. The diameters of uterus horns were 12x19 mm and 11x21 mm (left and right, respectively). The cervix was present but very small (size like that of a finger). Cytogenetic analyses showed a male constitution. Indeed, all cells we examined with CBA- and RBAbanding were 2n=64, XY (Figure 2B). No positive signals were detected when studying the results of the FISH-analysis with a bovine BAC-clone containing SRY (Figure 2C). The lack of the SRY gene was confirmed when molecular analyses were performed on genomic DNA of XY mare and control (fertile male and female) with a 795 bp fragment detecting the SRY gene (Figure 2D). DISCUSSION Numerical sex chromosome abnormalities have been correlated with sterility or hypofertility, especially in females, in several domestic species (Gustavsson 1980; Kent et al. 1986, 1996; Power 1986; Pailhoux et al. 1995; Makinen et al. 1999; Iannuzzi et al. 2000, 2001). Although one of two X-chromosomes is randomly inactivated, deleterious effects can arise when more than one X chromosome are found in cells of carriers. This can be explained by

3 402 iannuzzi, di meo, perucatti, spadetta, incarnato, parma, iannuzzi, ciotola, peretti, perrotta and di palo Fig. 1 CBA-banded metaphase plate of male horse affected by Klinefelter syndrome (2n=65, XXY). The two Xs and the Y-chromosome are indicated. the fact that some genes present in the X chromosome escape the inactivation process (reviewed by Tsuchiya et al. 2000). Klinefelter syndrome is the most common cause of hypogonadism and infertility in male humans (Powell 1999). In the human carriers, the origin of the extra chromosome arises from paternal (53 % of cases) and maternal (34 % of cases) meiosis I, maternal meiosis II (9% of cases) and postzygotic errors (3 %ofcases) (Powell, 1999). In horses, while several cases with chromosome mosaics (XX/XXY, XY/XXY, XX/XY/XXY, XXY/XX/XXYY) have been observed (reviewed in Makinen et al., 2000), only two pure cases of XXY stallions have been reported (Kubien et al. 1993; Makinen et al. 2000). In particular, the stallion described by Kubien et al. (1993) showed small and hypoplastic testes with no scrotum. The stallion studied by Makinen et al. (2000) was a taller horse, compared with the same males from the same breed and age, with soft testes and a smaller penis compared to the body size. After castration, testes were found to be appreciably smaller than those of normal stallions. Subsequent histological studies revealed few seminiferous tubules and no germinal epithelium. Both stallions were sterile. In our case the stallion showed normal body conformation, although it was taller than other stallions of the same age. However, penis, scrotum and testes, appeared slightly smaller compared to body size. Microscopic observation of semen revealed the absence of spermatozoa, probably due to alterations of testes as found by Makinen et al. (2000). Unfortunately, no subsequent analyses were performed when the stallion was mated. Sex reversal syndrome, known as Swyer s syndrome in humans, is the most complex of those involving sex chromosomes. The sex determining region (SRY) gene found in the Y chromosome is known to be responsible for testis formation. Hence, when Y-chromosome is present in the cells, male phenotype is observed even when more than one X is present in the cells. The only exception being the sex reversal syndrome which can be found in both XY females and XX males. This syndrome is not fully understood. Indeed, a mutation and/or deletion of SRY seems to be involved in this syndrome (Scherer et al. 1998; Koopman et al. 1991, 2001), but another member of the SRY-HMG box related family, SOX9 seems to play an important role in testis differentiation (reviewed in Pannetier et al., 2003). Recently, it was proved that the syndrome may also be related to changes in the protein encoded by the SRY gene. The altered protein has trouble entering the nucleus of fetal male gonadal cells and the genes which should be turned on by SRY to originate the testes remain off (Sreenivasan 2003). In our case, the results with both chromosomal FISH and molecular analysis revealed the lack of SRY gene, supporting the hypothesis of deletion as

4 clinical cytogenetics in horse 403 Fig. 2 (a) Posterior of mare affected by sex reversal syndrome. Notice the normal external genitalia. (b-c) Metaphase plates of mare affected by sex reversal syndrome and treated for CBA-banding (b) and FISH-mapping with a BAC-clone containing SRY gene (c). X and Y chromosomes are indicated. Notice the lack of hybridisation FITC-signals on the Y-chromosome (c). (d) Molecular analysis of SRY gene with a 795 bp fragment. Line M: Gene Ruler 100bp DNA ladder mix (Fermentas), line 1: normal male (SRY-positive), line 2: mare XY (SRY-negative), line 3: normal mare (SRY-negative), line 4: water.

5 404 iannuzzi, di meo, perucatti, spadetta, incarnato, parma, iannuzzi, ciotola, peretti, perrotta and di palo found in other previous XY mares (Abe et al. 1999; Pailhoux et al. 1995; Kent et al. 1996; Makinen et al. 2001; Vaughan et al. 2001; Bugno et al. 2003). Bugno et al. (2003) suggest that this deletion is probably due to two crossover events between X and Y during spermatogenesis of sire with the result that SRY translocated from Y to X and the Y chromosome is lacking this important gene. REFERENCES Abe S., Miyake Y-I., Kageyama S.I., Watanabe G., Taya L.K., Kawakura K., 1999 Deletion of the SRY region on the Y chromosome detected in a case of equine gonadal hypoplasia (XY female) with abnormal profiles. Equine Vet. J., 31: Bugno M., Klukowska J., Slota E., Tischner M., Switonski M., 2003 A sporadic case of the sex-reversal mare (64,XY; SRY-negative: molecular and cytogenetic studies of the Y chromosome. Therigenology, 59(7): Chowdhary B.P., Rausepp T., 2000 Cytogenetics and physical gene map. In CAB international The genetics of the horse (Bowling AT, Ruvinsky A, eds), p Hasegawa T., Ishida M., Harigaya T., Sato F., Ishida N., Mukoyama H., 1999 Linear SRY transcript in equine testis. J. Vet. Med. Sci., 61(1): Gustavsson I., 1980 Chromosome aberrations and their influence on the reproductive performance of domestic animals-a review. Z. Tierzuchtg. Zuchtgsbiol., 97: Iannuzzi L., 2003 Methodologies applied on domestic animal chromosomes. From Methods in Molecular Biology, vol. 240: Artificial Mammalian Chromosomes, V. Sgaramella, S. Eridani (eds), Humana Press, Totowa, NJ, p Iannuzzi L., Di Meo G.P., Perucatti A., Di Palo R., Zicarelli L., 2001a 50, XY gonadal dysgenesis (Swier s syndrome) in a female river buffalo (Bubalus bubalus). Vet. Record., 148: Iannuzzi L., Molteni L., Di Meo G.P., De Giovanni A., Perucatti A., Succi G., Incarnato D., Eggen A., Cribiu E.P., 2001b A case of azoospermia in a cattle bull carrying an Y-autosome reciprocal translocation. Cytogenet. Cell Genet., 95: Iannuzzi L., Di Meo G.P., Perucatti A., Zicarelli L., 2000 A case of sex chromosome monosomy (2n=49,X0) in the river buffalo (Bubalus bubalis). Vet. Record, 147: Iscnh, 1997 International System for Cytogenetic Nomenclature of the domestic Horse. Bowling A.T., Breen M., Chowdhary B.P., Hirota K., Lear T., Millon L.V., Ponce De Leon F.A., Raudsepp T., Stranzinger G. (Committee). Chrom. Res. 5: Kent M.G., Meallem A., First NL., 1996 Molecular etiology of sex reversal in the horse. Cytogenet. Cell Genet., 74:234. Kent M.G., Shoffner R.N., Buoen L., Weber A.F., 1986 XY sex-reversal syndrome in the domestic horse. Cytogenet. Cell Genet., 42: Koopman P., Gubbay J., Vivian N., Goodfellow P., Lovell-Badge R., 1991 Male development of chromosomally female mice transgenic for Sry. Nature, 351: Koopman P., Bullejos M., Bowles J., 2001 Regulation of male sexual development by Sry and Sox9. J. Exp. Zool., 290: Kubien E.M., Pozor M.A., Tischener M., 1993 Clinical, cytogenetic and endocrine evaluation of a horse with a 65,XXY karyotype. Equine Vet. J., 25: Makinen A., Hasegawa T., Makila M., Katila T., 1999 Infertility in two mares with XY and XXX sex chromosomes. Equine Vet. J.; 31: Makinen A., Suojala L., Niini T., Katila T., Tozaki T., Miyake Y-I., Hasegawa T., 2003 X chromosome detection in a X0 mare using a human X paint probe, and PCR detection of SRY and amelogenin genes in three XY mares. Equine Vet. J., 33: Makinen A., Katila T., Anderson M., Gustavsson I., 2000 Two sterile stallions with XXY-syndrome. Equine Vet. J., 32: Pailhoux E., Cribiu E.P., Parma P., Cotinot C., 1995 Molecular analysis of an XY mare with gonadal dysgenesis. Hereditas, 122: Pannetier M., Servel N., Cocquet J., Besnard N., Cotinot C., Pailhoux E., 2003 Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals. Cytogenet. Genome Res., 101: Powell C., 1999 Sex chromosomes and sex chromosome abnormalities. In The principles of clinical cytogenetics, Gersen SL, Keagle MB, eds. Humana Press, p Power M.M., 1986 XY sex reversal in a mare. Equine Vet. J., 18: Scherer G., Held M., ErdeL M., Meschede D., Horst J., Lesniewicz R., Midro A.T., 1998 Three novel SRY mutations in XY gonadal dysgenesis and the enigma of XY gonadal dysgenesis cases without SRY mutations. Cytogenet. Cell Genet., 80: Sreenivasan A., 2003 In the sex reversal, protein deterred by nuclear barrier. Science, 302: Thrommershausen-Bowling A., Millon L., Hughes J.P., 1987 An update of chromosomal abnormalities in mares.j.reprod. Fert., Suppl 35: Tsuchiya K.D., Willard H.F., 2000 Chromosomal domains and escape from X inactivation: comparative X inactivation analysis in mouse and human. Mammal. Genome, 11: Vaughan L., Schofield W., Ennis S., 2001 SRYnegative sex reversal in a pony: a case report. Theriogenology, 55: