Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:65-68
Two Cases of Infertile Bitches With 78,XX/77,X Mosaic Karyotype: A Need for Cytogenetic Evaluation of Dogs With Reproductive Disorders
From the Department of Genetics and Animal Breeding (Switonski, Szczerbal, and Grewling) and the Department of Veterinary Agriculture (Antosik), August Cieszkowski Agricultural University of PoznÄn, Wolynska 33, 60-637 PoznÄn, Poland; the Department of Animal Reproduction, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Wroclaw, Poland (Nizanski); and the Center for Veterinary Science, Madingley Road, University of Cambridge, Cambridge, United Kingdom (Yang).
Address correspondence to Marek Switonski at the address above, or e-mail: switonsk{at}jay.au.poznan.pl.
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Abstract |
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A mosaic karyotype 78,XX/77,X was found in two infertile purebred dogs. Cytogenetic investigations were carried out on a large number of metaphase spreads (220 and 473) with the use of conventional Giemsa staining, C-banding, and chromosome painting approaches. The frequencies of the monosomic spreads (77,X) were low: 5.3% and 5.6%. We recommend that at least 90 metaphase spreads be analyzed to allow possible detection of low-level XX/X mosaicism, and we discuss the need for cytogenetic evaluation of dogs with reproductive disorders.
During recent years, cytogenetic studies of dog chromosomes have focused on establishing the standard karyotype and physical localization of the marker loci (Breen et al. 2001a,b). This has resulted in very rapid progress of the canine marker genome map, which has facilitated identification of genes responsible for hereditary diseases (Patterson 2000). On the other hand, clinical cytogenetic studies of malformed and infertile dogs have been rather poorly developed. The small number of cytogenetic reports on the dog has been caused in part by difficulty in recognizing autosomes by the use of banding techniques (Switonski et al. 1996). It should be emphasized, though, that identification of the sex chromosomes is easy due to their biarmed morphology and peculiar C-banding patterns of the X chromosome (interstitial band in the q arm).
Our aim in this study was cytogenetic evaluation of two infertile female dogs. Blood samples were provided by veterinary practitioners.
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Materials and Methods |
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Case 1
A 3-year-old Munsterlander bitch was subjected for cytogenetic analysis due to infertility. The appearance of her external reproductive organs was normal; however, endoscopic inspection of the vagina revealed an elongated vertical septum extending from the region of the urethral orifice cranially to the uterine cervix. The size and conformation of the vulva, vaginal vestibule, clitoris, and urethral papilla were typical for the breed. Ultrasound examination of the cranial part of the genital tract with the use of a 7.5 MHz transducer did not reveal any abnormalities of the ovaries or uterus. The course and clinical signs of proestrus and estrus were physiological each time. The bitch allowed normal copulation with typical tie in two standing heats and was bred by two fertile stud dogs in two consecutive estrus cycles.
Case 2
A 5-year-old bearded collie female was referred for cytogenetic studies due to infertility. Her external genitalia were normal, but her estrus cycles were irregular and were poorly manifested. The bitch was unsuccessfully mated two times with a fertile male and inseminated four times.
In both dogs, the optimal time for breeding was estimated by cytological examination of vaginal smears and, in case 1, by monitoring the peripheral serum progesterone concentration at 2-day intervals. The pregnancy diagnosis by ultrasound examination was performed 28 and 35 days after the first breeding. The examinations did not show any fetuses and the bitches did not bear any puppies. Sires and dams of the studied bitches demonstrated normal reproductive performance.
Chromosome spreads were obtained from routine in vitro cultured lymphocytes. Evaluation of the chromosome sets was carried out with the use of conventional Giemsa staining, CBG banding, and chromosome painting with the canine whole X chromosome painting probe (Yang et al. 1999). Standard protocols for C-banding and fluorescent in situ hybridization (FISH) were applied. International canine chromosome nomenclature was applied (Switonski et al. 1996). Microscopic evaluation was carried out under a fluorescent microscope (Nikon Eclipse 600) equipped with a cooled CCD digital camera and Lucia software.
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Results and Discussion |
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A total of 220 chromosome spreads originating from case 1 were analyzed, and among them 5.3% had only a single X chromosome (Table 1). The monosomic spreads were detected on Giemsa-stained and C-banded spreads and also after application of the X chromosome paint (Figure 1a). The karyotype was designated as a mosaic 78,XX/77,X.
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Cytogenetic evaluation of case 2 also revealed the mosaic karyotype—78,XX/77,X. Among 473 analyzed spreads, 5.6% were monosomic (Table 1). The cytogenetic analysis was mainly carried out on the FISH-painted spreads, but 77,X spreads were also identified among Giemsa-stained ones (Figure 1b).
Identification of the two mosaic 78,XX/77,X bitches indicates that this abnormality can be an important genetic factor contributing to canine infertility. These results were unexpected, since only three other cases—two pure X monosomy and one XX/X mosaicism—have been diagnosed in the dog (Table 2). Other sex chromosome aneuploids have been described in the dog. There are two reports of X trisomy (Johnston et al. 1985; Switonski et al. 2000) and seven cases of XXY trisomy (for a review, see Mellink and Bosma 1989).
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The cases reported in this study should be classified as low-level mosaicism, since the monosomic cell line (77,X) was represented by approximately 5% of the studied lymphocytes. Diagnosis of these cases was possible because a large number of spreads were analyzed (220 and 473, respectively). From the mosaicism tables (Hook 1977) it is known that to exclude mosaicism at the level of 5%, with 99% confidence, one has to analyze at least 90 cells. If all of them have the same karyotype, then the mosaicism can be excluded. The number of analyzed spreads in this study is sufficient to exclude mosaicism at 3% (case 1) and 1% (case 2).
Low-level mosaicism is a well-known phenomenon. To detect such cases, a large number of evaluated metaphase spreads are needed, and thus application of the FISH technique is very helpful. Low-level mosaicism XX/X, detected by the chromosome painting approach, was recently described in infertile mares (Wieczorek et al. 2001). So-called hidden mosaicism has been described in human patients with Turner syndrome (Fernandez-Garcia et al. 2000). They detected low-level mosaicism in patients previously classified as nonmosaic Turner syndrome (45,X). The application of the FISH technique revealed the presence of XX cells with a low frequency, from 0.2% to 6%; on the other hand, extensive studies of the human Turner syndrome revealed a wide range in monosomic cell line frequencies in different tissues originating from the same patient (Held et al. 1992). In some cases, they identified the X monosomic cell line with a low frequency (less than 5%) in lymphocytes and with a very high frequency (up to 99%) in fibroblasts. Thus the reversed situation was found. This clearly shows that the frequency of spreads with X monosomy in lymphocytes may not be representative for the entire body.
X monosomy causes a so-called Turner syndrome in women. The major symptom of this disorder is atresia of the ovarian follicles, causing infertility and some morphological malformations including growth retardation, congenital heart disease, webbed neck, and hearing disorders (Hall and Gilchrist 1990). Because of the very small number of diagnosed cases of X monosomy in the dog (Table 2), our knowledge about the morphological abnormalities caused by this status is very limited. In our study, the only malformation identified was vaginal septum (case 1). It seems rather unlikely that this feature is related to the XX/XO mosaic status.
From a clinical point of view, it is important to mention that in women with Turner syndrome, a relation between the presence and degree of mosaicism and phenotype is observed. For instance, sporadic pregnancies occur more frequently in women with the mosaic karyotype than in nonmosaic ones (Tarani et al. 1998). In some cases of XX/X mosaicism in female dogs, subfertility may occur instead of infertility, and this may be supported by the observation of a normal course of estrus in case 1. It should be also emphasized that women with the mosaic karyotype have fewer hearing and cardiac problems than do pure monosomics. The clinical importance of detection of low-level mosaicism as a prognostic tool was presented by Hanson et al. (2001); the authors applied the FISH technique on interphase nuclei to detect the presence of X and Y chromosome material in 53 women with Turner syndrome. The FISH technique revealed a much higher proportion of the patients (70%) with X-chromosomal mosaicism then was previously detected by conventional chromosome banding techniques (45%). Moreover, for 15% of the patients, Y chromosome material was also detected. Of interest is that in the case of X monosomy in a Doberman pinscher bitch, the presence of the cell line carrying the Y chromosome was postulated (Giger et al. 1989).
Our study shows that the incidence of 78,XX/77,X mosaicism among infertile and subfertile female dogs may be much higher than could be predicted from the available published reports. Thus this abnormality, as well as sex-reversal syndrome (78,XX; SRY-negative) (Meyers-Wallen et al. 1999), may be an important genetic cause of female dog reproductive disorders. Cytogenetic investigations of infertile and subfertile dogs are needed to analyze a large number of metaphase spreads (minimum 90–100) for a proper diagnosis.
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Acknowledgments |
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This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
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Footnotes |
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Corresponding Editor: Urs Giger
Received July 15, 2002
Accepted September 19, 2002
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References |
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Breen M, Jouquand S, Renier C, Mellersh CS, Hitte C, Holmes NG, Cheron A, Suter N, Vignaux F, Bristow AE, Priat C, McCann E, Andre C, Boundy S, Gitsham P, Thomas R, Bridge WL, Spriggs HF, Ryder EJ, Curson A, Sampson J, Ostrander EA, Binns MM, Galibert F, 2001a. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11:1784-1795.
Breen M, Switonski M, Binns MM, 2001b. Cytogenetics and physical chromosome maps. In: The genetics of the dog (Ruvinsky A and Sampson J, eds). Wallingford, UK: CAB International; 299–328.
Fernandez-Garcia R, Garcia-Doval S, Costoya S, Pasaro E, 2000. Analysis of sex chromosome aneuploidy in 41 patients with Turner syndrome: a study of "hidden" mosaicism. Clin Genet. 58:201-208.[CrossRef][ISI][Medline]
Giger U, Meyers-Wallen VN, Patterson DF, 1989. Mosaicism in a dog rather than simple X-chromosomal monosomy [letter]. J Vet Intern Med. 3:245.[Medline]
Hall JG, Gilchrist DM, 1990. Turner syndrome and its variants. Pediatr Clin North Am. 37:1421-1440.[ISI][Medline]
Hanson L, Bryman I, Barrenas M-L, Janson P-O, Wahlstrom J, Albertsson-Wikland K, Hanson C, 2001. Genetic analysis of mosaicism in 53 women with Turner syndrome. Hereditas. 134:153-159.[CrossRef][ISI][Medline]
Held KR, Kerber S, Kaminsky E, Sungh S, Goetz P, Seemanova E, Goedde HW, 1992. Mosaicism in 45,X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes? Hum Genet. 88:288-294.[ISI][Medline]
Hook EB, 1977. Exclusion of chromosomal mosaicism: tables of 90%, 95%, and 99% confidence limits and comments on use. Am J Hum Genet. 29:94-97.[ISI][Medline]
Johnston SD, Buoen LC, Weber AF, Madl JE, 1985. X trisomy in an Airedale bitch with ovarian dysplasia and primary anestrus. Theriogenology. 24:597-606.
Lofstedt RM, Buoen LC, Weber AF, Johnston SD, Huntington A, Concannon PW, 1992. Prolonged proestrus in a bitch with X chromosomal monosomy (77,XO). J Am Vet Med Assoc. 200:1104-1106.[ISI][Medline]
Mayenco-Aguirre AM, Padilla JA, Flores JM, Daza MA, 1999. Canine gonadal dysgenesis syndrome: a case of mosaicism (77,XO-78,XX). Vet Rec. 145:582-584.[ISI][Medline]
Mellink CNM, Bosma AA, 1989. The karyotype of the dog (Canis familiaris L.). In: Cytogenetics of animals (Halnan CRE, ed). Wallingford, UK: CAB International; 151–158.
Meyers-Wallen VN, Schlafer D, Barr I, Lovell-Badge R, Keyzner A, 1999. Sry-negative XX sex reversal in purebred dogs. Mol Reprod Dev. 53:266-273.[CrossRef][ISI][Medline]
Patterson DF, 2000. Companion animal medicine in the age of medical genetics. J Vet Intern Med. 14:1-9.[CrossRef][ISI][Medline]
Smith FW, Jr, Buoen LC, Weber AF, Johnston SD, Randolph JF, Waters DJ, 1989. X-chromosomal monosomy (77,XO) in Doberman pinscher with gonadal dysgenesis. J Vet Intern Med. 3:90-95.[ISI][Medline]
Switonski M, Godynicki S, Jackowiak H, Pienkowska A, Turczuk-Bierla I, Szymas J, Golinski P, Bereszynski A, 2000. X trisomy in an infertile bitch: cytogenetic, anatomic and histologic studies. J Hered. 91:149-150.
Switonski M, Reimann N, Bosma AA, Long S, Bartnitzke S, Pienkowska A, Moreno-Milan MM, Fischer P, 1996. Report on the progress of standardization of the G-banded canine (Canis familiaris) karyotype. Chromosome Res. 4:306-309.[CrossRef][ISI][Medline]
Tarani L, Lampariello S, Reguso G, Colloridi F, Pucarelli I, Pasquino AM, Bruni LA, 1998. Pregnancy in patients with Turner's syndrome: six new cases and review of literature. Gynecol Endocrinol. 12:83-87.[ISI][Medline]
Wieczorek M, Switonski M, Yang F, 2001. A low-level chromosome mosaicism in mares, detected by chromosome painting. J Appl Genet. 42:205-209.[Medline]
Yang F, O'Brien PCM, Milne BS, Graphodatsky AS, Solanky N, Trifonov V, Rens W, Sargan D, Ferguson-Smith MA, 1999. A complete comparative chromosome map of the dog, red fox, and human and its integration with canine genetic maps. Genomics. 62:189-202.[CrossRef][ISI][Medline]
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