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Multiple Congenital Genitourinary Anomalies in a Polled Goat WILLIAM W. KING, DVM, PHD, DIPLOMATE, ACLAM,1,2* MELVIN E. YOUNG,1 AND M. EUGENE FOX, DVM3 A 1-day-old, Toggenburg/Nubian crossbred goat of polled parentage was referred for necropsy because of a large (diameter, 5 cm) bladder-like mass protruding from the perineal midline and difficult urination. Differential diagnoses included cutaneous cyst, ectopic urinary bladder, and urethral diverticulum/dilatation. Several genitourinary aberrations were noted. A second, smaller (diameter, 1 cm), more distal cystic structure was adjacent to an ambiguous prepuce. Testicles were discovered within a constricted, subcutaneous space near the inguinal canals. A rudimentary penis was located dorsal to the penile urethra with no appreciable urethral process. A tiny external urethral orifice was discerned only after liquid was injected into the lumen of the cystic structures, confirming their identity as urethral dilatations. The dilatations were separated by a constricting band of fibrous tissue. No other significant findings were detected. This case illustrates a combination of congenital anomalies including bilateral cryptorchidism with scrotal absence, segmental urethral hypoplasia, and urethral dilatation, most likely associated with the intersex condition seen in polled breeds. The continued production and use of small ruminants as animal models demands the prompt recognition of congenital anomalies. This case also exemplifies the precautions required when breeding goats with polled ancestry. The domestic goat (Capra hircus) has historically served and continues to play an important role in biomedical research (1). Many small breeds are available, facilitating common laboratory animal husbandry practices. Because of their large, easily accessed jugular veins, goats frequently are used in the production of antiserum. Along with sheep (Ovis aries), these small ruminants are common subjects in reproductive studies. Because of their natural affinity for caprine arthritis-encephalitis virus, goats also are used to investigate the pathogenesis of lentivirus infection. This species has been specifically recommended as a spontaneous animal model for the following human diseases: congenital hypothyroidism (2), neuritic plaques (secondary to scrapie; 3), pulmonary adenomatosis (jaagziekte, or alveolar cell carcinoma; 4), amelia, or congenital absence of limbs (5), and glomerulonephritis (6). Many conditions are known to be inherited in various goat breeds, including afibrinogenemia (Saanen), beta-mannosidosis (Nubian), bipartite scrotum (Angora), brachygnatha or abnormally shortened mandible, cryptorchidism (Angora), excessive facial hair (Angora), gynecomastia or excessive development of male mammary glands, hereditary goiter (Dutch), inherited abortion (South African Angora), the spasm-inducing disorder myotonia congenita, recessive atrichosis or hairlessness, Robertsonian translocation, short tendons (Australian Angora), sperm granulomas, supernumerary teats, and testicular hypoplasia (7). Although numerous congenital disorders have been reported in goats, the presence of multiple anomalies in a single case is rather rare (8). This case report describes a combination of abnormalities most likely associated with the intersex condition in a polled kid. Case History A 1-day-old goat was referred for necropsy because of dysuria and a large bladder-like mass extending from the ventral perineal midline. The animal was examined by the referring veterinarian; euthanasia was elected and performed on-site. The kid was one of a pair of twins from a polled Toggenburg dam and polled 1 Research Service (151), Department of Veterans Affairs, Edward Hines Jr. VA Hospital, Hines, Illinois 60141; 2Department of Pathology, Loyola University Medical Center, Maywood, Illinois 60153; 3House-Call Veterinary Service, Lockport, Illinois 60441 * Corresponding author: William W. King, DVM, PhD, Diplomate, ACLAM, Office of Research Services, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Volume 41, No. 5 / September 2002 Nubian/Toggenburg sire. The owner reported that the doe had completed a normal gestation period on a diet of natural grass/ alfalfa hay and water. The surviving twin appeared to be a normal male. Upon initial gross examination, the kid appeared in good nutritional status. The prepuce demonstrated shared characteristics of female and male, with the preputial opening located intermediately between that expected of a male or female. The prepuce was characterized as a small (approximately 1 cm) opening in the ventral abdominal skin, exposing a slightly globoid, reddened mucous membrane with little recognizable structure. A small (approximately 0.2 × 1.0 cm) cartilaginous structure was palpable dorsal to the prepuce. No urethral process was present in the prepuce, and no other external genitalia were identifiable. A 5-cm, vascularized, bladder-like mass extended from the ventral perineal midline (Fig. 1). A similar, smaller (approximately 1 cm) structure was located adjacent to the prepuce. The two were separated by a constricting band of fibrous tissue just proximal to the area of the preputial urethra. The surface of these thin-walled structures was smooth with no evidence of hair or other skin adnexa. A tiny external urethral orifice was recognized only after liquid was injected into, and expressed from, the bladder-like mass. Dissection revealed that the lumen of these structures was confluent with the pelvic urethra. Upon further examination, testicles were discovered within a short inguinal canal confined within the subcutis (Fig. 2). Grossly, the testes appeared normal, although the ductus deferens appeared distended throughout the length of the colliculus seminalis to the epididymis. No indication of a scrotum was evident. No rudimentary ovarian or uterine structures were located, and no other lesions were appreciated grossly. Differential diagnoses for the larger bladder-like structure included cutaneous cyst, ectopic urinary bladder, and urethral dilatation or diverticulum. Dissection of the perineum confirmed that both structures constituted dilatations/diverticula of the perineal urethra. Further exploration identified the urethral lumen from the urinary bladder to the small external urethral orifice (Fig. 3). Gross observations were consistent with the diagnoses of bilateral cryptorchidism, segmental urethral hypoplasia, and urethral diverticula. The preputial ambiguity strongly suggested a genetic condition involving reproductive development. In neonatal ruminants, CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science 39 Figure 3. Dissected genitourinary tract of affected goat. A catheter has been inserted through the urethra into the urinary bladder. A, external urethral orifice; B, large urethral diverticulum; C, seminal colliculus; D, small urethral diverticulum. Discussion Figure 1. External appearance of anomalies. Note the two ventral midline cyst-like masses and the ambiguous prepuce. Figure 2. Location of cryptorchid testicle (A) in subcutaneous inguinal canal (B, vaginal ring). the distal aspect of the penis, including the urethral process in goats, is adhered to the prepuce (9). However, the testicles normally are descended at birth into a well-developed scrotum (7). In this case, only a rudimentary penile structure was identifiable, the prepuce was located in an intermediate position, and, unlike in the surviving twin male, no scrotum was evident. In light of these findings, the known parentage, and the similarities with other clinical reports, the presumptive etiology for the anomalies seen in this case is the intersex condition seen in polled breeds. Limitations in the tissues available for additional diagnostic evaluation made definitive differentiation from freemartinism impossible. 40 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science Congenital abnormalities. Developmental anomalies may result from both genotypic and environmental influences, or a combination of both, with an interruption of the “temporal-spatial requirements of development” (10). Congenital or hereditary abnormalities of the urethra occur infrequently in all animal species and include malformations of the urethral wall and orifice (e.g. hypospadias, epispadias with exstrophy or eversion of the bladder), urethral aplasia and agenesis, ectopic urethra, urethral duplication, and urethrorectal or urethrovaginal fistula (11, 12). In goats, inherited anomalies associated with the distal urinary tract and/or reproductive tract include bipartite scrotum and cryptorchidism in Angora goats, testicular hypoplasia, sperm granulomas, and intersex (7). Reports of urethral diverticula in neonatal goats are uncommon. In one description, a 2-day-old goat of unspecified breed presented for urinary obstruction with a urethral diverticulum (confirmed via positive contrast urethrography), bipartite scrota, and preputial hypoplasia with an imperforate urethral process (8). Although no etiology was determined, surgical correction (urethrostomy, obliteration of the diverticula, and castration) was effective. In a report of 11 goats between 2-6 months of age that presented with continuous dribbling of urine, congenital urethral diverticula were discovered and successfully treated by surgical resection. Ten of the kids, which were of unstated breed, also had scrotal anomalies, including bipartite scrotum and monorchidism (13). Although the term “pseudohermaphrodite” has been used to describe animals retaining the gonads of one gender with sex characteristics of the other, such as genotype or reproductive morphology (14), the preferred term for gender inconsistencies in goats is “intersex” (7). This condition results from sex chromosomal abnormalities and has been defined as “ambiguity in the structure of the gonads, reproductive tract, or external genitals” (14). The disorder is strongly associated with the polled condition in goat breeds of western European descent (Saanen, Alpine, and Toggenburg) and is less commonly found with freemartinism (15, 16). There have been several descriptive surveys of the intersex condition in dairy goats. Hamerton and colleagues examined 35 cases of polled intersexuality and found that the vast majority (97%) of cases were genetically female (60,XX) with mixed male phenotype and that there was a low percentage (3%) of freemartins (60,XX/60,XY) (15). In 1943, Eaton described 29 cases of “pseudohermaphroditism,” classifying each into four “female-predominant” and four “male-predominant” categories Volume 41, No. 5 / September 2002 based on external anatomy, with six additional categories based on internal anatomy (17). The case we describe shared many characteristics of the class 2 male-predominant type, with the exception of the urethral opening at the tip of the genitalia and urethral diverticula at the expanse of the perineal raphe (central midline). Also of interest, Eaton noted that 29% of the male-predominant cases presented with hypospadias, or ventral opening of the penile urethra. The occurrence of the urethral diverticula in this case may represent an anomalous closure of the ventral urethral wall, similar to hypospadias, or be associated with agenesis/hypoplasia of the urethral opening. Other findings reported to occur in conjunction with the intersex condition include cryptorchidism, in which the testes typically are retained within the abdomen but may be partially or fully descended (16). A more recent report involving an 18-day-old intersex FrenchAlpine/Nubian goat had remarkable similarities to the case described in the present report (18). This kid presented with a ventral midline urethral dilatation associated with a hypoplastic distal urethra at the prepuce. The urethra was found to be distinctly separate from a rudimentary penis; surgical correction and urethrostomy was performed. During an exploratory abdominal surgery for gonadectomy, testicles were located adjacent to each kidney. Blood karyotype analysis revealed that the goat was genotypically female (60,XX). Genetic anomalies. Gender development results from complex interactions of sex- and autosomal-linked genes. The SRY (sex-determining region Y) gene directs mammalian embryos to develop testes, which subsequently support the male phenotype by producing Mullerian inhibiting proteins and testosterone (19, 20). This cascade of genetic events is not simple, however, as is illustrated in the intersex condition seen in polled goats. The interaction between hornlessness and intersex in genetically female (XX) goats, first suggested by Asdell in 1944 (21), has been dubbed the Polled Intersex Syndrome (PIS) and is thought to be caused by an interference with the development of female characteristics by aberrant expression of male-promoting gene products (7). Phenotypically, the mutation (P), which is either a single pleiotropic gene or two intimately linked genes, expresses polledness in an autosomal dominant fashion and results in sex-reversal in females as an autosomal recessive trait (22). The mutation partially simulates the Y chromosome, encouraging the development of testicular tissue by generating H-Y antigen (16, 23, 24). Interestingly, the PIS XX intersex is SRY-negative, a fact that supports theories of autosomal gene involvement in male gender development independent the Y-chromosome or of X-Y or autosomal-Y translocation of SRY genes (25, 26). The mutation causing PIS recently has been identified as an 11.7kb deletion on chromosome 1 (20, 27, 28). Females that are homozygous for the polled gene (PP) will become infertile intersex (7); fertile polled females therefore must be heterozygous for the polled gene (Pp) (16). Intersex secondary to freemartinism is another possibility in the described case. The pathogenesis of this condition involves the sharing of cells and hormones between a male and female fetus following placental fusion and anastomosis, resulting in chimerism in the female twin (16). Karyotype analysis generally is used to differentiate polled intersex (genetically female, XX) from freemartinism (chromosomal chimerism, XX/XY). This assay can be performed on lymphocytes from whole blood or cultured renal or pericardial fibroblasts (10). Several authors have described the use of polymerase chain reaction (PCR) techniques for determining sex in goats (29, 30). Examination of fresh tissue with either PCR-based assays or Southern blot for the presence of the Y-chromosome-associated Zfy and Sry regions can differentiate polled intersex from freemartin Volume 41, No. 5 / September 2002 Figure 4. Breeding schemes associated with the intersex condition. Note that a homozygous polled doe (PP) is infertile and therefore not shown. (Modified from Smith and Sherman, 1994.) chimerism (25, 26). Unfortunately, appropriate tissues were not archived from the described goat to perform these confirmatory steps. However, the prevalence of placental fusion is much lower in goats than in cattle, making freemartinism much less common in goats (7). Reproductive management. Institutions and animal breeders producing goats may find the congenital lack of horns a desirable characteristic with practical and humane advantages over surgical dehorning or disbudding. However, there are several problems associated with the polled condition. An understanding of the mechanisms of transmission of PIS therefore is extremely important when designing a production program. Figure 4 illustrates the breeding crosses that may result in intersex offspring. The intersex condition in European goats can be avoided by ensuring that one parent was born with horns (genotypically horned), that is, homozygous recessive for the polled gene (p). This precaution will prevent any female offspring from expressing the intersex associated with the homozygous dominant polled gene (P) (7). Note that a central whorl of hair on the dorsal cranium is present in polled goats; two whorls overlying the area of horn growth is present in disbudded kids (7). In programs designed to increase the number of polled offspring, the goals of increasing fertility and decreasing the potential for intersexes often conflict. To maximize reproductive output, breeding horned bucks (pp) to fertile (therefore heterozygous, Pp) polled does, which often produce more offspring including twins and triplets (16), will result in only 50% of the offspring being polled but with no possibility of PIS females. Although there are disadvantages in using homozygous polled bucks (PP), which frequently develop sperm granulomas resulting in obstruction of the head of the epididymis and testicular hypoplasia (7, 16), the cross of such a sire with a genotypically horned doe (pp) results in 100% heterozygous polled offspring, also with no potential for producing polledassociated intersexes. Crossing a polled buck (regardless of genotype) with a doe born horned (pp) will result in a 6/8 (75.0%) probability of polled offspring with no possibility of PIS females. Breeding a polled buck (again, regardless of genotype) with a polled doe (Pp) will result in a 7/8 (87.5%) probability of polled offspring but a concurrent 3/8 (37.5%) chance of intersex in female kids. Alternatively, breeding schemes can be designed to specifically produce PIS animals for use as models of the human XX-male intersex condition. In humans, conditions leading to intersex may occur at any time during intrauterine development and may lead to a spectrum of consequences ranging from overt anatomical and physiological anomalies to more elusive abnormalities not appreciated until sexual maturity (31). Although considerable work has focused on the appropriate timing of treatment, intersexuality continues to result in a significant incidence in gender identity disorders (32). The presentation of such infants produces a two-fold crisis: 1) challenging medical and CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science 41 surgical interventions, and 2) extensive sociological and psychological adjustment (33, 34). Of particular interest, it has been recently suggested that the PIS gene is homologous to the locus linked to blepharophimosis ptosis epicanthus inversus syndrome (BPES), a condition associated with ovarian dysfunction and epidermal thickening of the eyelid in affected humans (27, 35). Intentional production of intersex goats is relatively straightforward—the cross of a fertile polled female goat (Pp) with a homozygous polled male (PP) will result in a 50% chance of PIS in female offspring. Thus, the polled goat will continue to advance the knowledge of mammalian XX-male sex reversal and other intersex conditions as well as the underlying intricate interactions involved in gender development. This report describes an interesting combination of congenital anomalies in a polled goat kid, including bilateral inguinal cryptorchidism and scrotal aplasia, segmental urethral hypoplasia, and urethral diverticula. These lesions most likely were associated with the intersex syndrome recognized in various polled European breeds. Expression of gene-associated disorders will continue to become more prevalent should breeding programs for goats become more intense. Proper design of breeding programs can minimize or maximize the probability of producing intersex animals. Acknowledgments This material is based upon work supported by the Office of Research and Development of the Department of Veterans Affairs. An abstract of this report was presented at the 1998 National Meeting of the American Association for Laboratory Animal Science in Cincinnati, Ohio. References 1. Brooks, D. L., P. C. Tillman, and S. M. Niemi. 1984. Ungulates as laboratory animals, p. 273-295. In J. G. Fox, B. J. 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Fine mapping suggests that the goat polled intersex syndrome and the human blepharophimosis ptosis epicanthus syndrome map to a 100-kb homologous region. Genome Res. 10:311-318. Volume 41, No. 5 / September 2002