Multiple Congenital Genitourinary Anomalies in a Polled

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 . 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. were discovered within a con- stricted, 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 with scrotal absence, segmental urethral hypoplasia, and urethral dilatation, most likely associated with the 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 with polled ancestry.

The domestic goat (Capra hircus) has historically served and Nubian/Toggenburg sire. The owner reported that the doe had continues to play an important role in biomedical research (1). completed a normal gestation period on a diet of natural grass/ Many small breeds are available, facilitating common labora- alfalfa hay and water. The surviving twin appeared to be a nor- tory animal husbandry practices. Because of their large, easily mal male. accessed jugular veins, goats frequently are used in the produc- Upon initial gross examination, the kid appeared in good tion of antiserum. Along with sheep (Ovis aries), these small nutritional status. The prepuce demonstrated shared charac- ruminants are common subjects in reproductive studies. Be- teristics of female and male, with the preputial opening located cause of their natural affinity for caprine arthritis-encephalitis intermediately between that expected of a male or female. The virus, goats also are used to investigate the pathogenesis of prepuce was characterized as a small (approximately 1 cm) lentivirus infection. This species has been specifically recom- opening in the ventral abdominal skin, exposing a slightly mended as a spontaneous animal model for the following globoid, reddened mucous membrane with little recognizable human diseases: congenital hypothyroidism (2), neuritic structure. A small (approximately 0.2 × 1.0 cm) cartilaginous plaques (secondary to scrapie; 3), pulmonary adenomatosis structure was palpable dorsal to the prepuce. No urethral pro- (jaagziekte, or alveolar cell carcinoma; 4), amelia, or congeni- cess was present in the prepuce, and no other external genitalia tal absence of limbs (5), and glomerulonephritis (6). were identifiable. Many conditions are known to be inherited in various goat A 5-cm, vascularized, bladder-like mass extended from the breeds, including afibrinogenemia (Saanen), beta-mannosidosis ventral perineal midline (Fig. 1). A similar, smaller (approxi- (Nubian), bipartite (Angora), brachygnatha or abnor- mately 1 cm) structure was located adjacent to the prepuce. The mally shortened mandible, cryptorchidism (Angora), excessive two were separated by a constricting band of fibrous tissue just facial hair (Angora), gynecomastia or excessive development of proximal to the area of the preputial urethra. The surface of male mammary glands, hereditary goiter (Dutch), inherited these thin-walled structures was smooth with no evidence of hair abortion (South African Angora), the spasm-inducing disorder or other skin adnexa. A tiny external urethral orifice was recog- myotonia congenita, recessive atrichosis or hairlessness, nized only after liquid was injected into, and expressed from, Robertsonian translocation, short tendons (Australian Angora), the bladder-like mass. Dissection revealed that the lumen of these sperm granulomas, supernumerary teats, and testicular hypo- structures was confluent with the pelvic urethra. plasia (7). Although numerous congenital disorders have been Upon further examination, testicles were discovered within a reported in goats, the presence of multiple anomalies in a single short confined within the subcutis (Fig. 2). Grossly, case is rather rare (8). This case report describes a combination the testes appeared normal, although the ductus deferens ap- of abnormalities most likely associated with the intersex condi- peared distended throughout the length of the colliculus tion in a polled kid. seminalis to the . No indication of a scrotum was evi- dent. No rudimentary ovarian or uterine structures were located, Case History and no other lesions were appreciated grossly. A 1-day-old goat was referred for necropsy because of dysuria Differential diagnoses for the larger bladder-like structure and a large bladder-like mass extending from the ventral perineal included cutaneous cyst, ectopic urinary bladder, and urethral midline. The animal was examined by the referring veterinar- dilatation or diverticulum. Dissection of the perineum confirmed ian; euthanasia was elected and performed on-site. The kid was that both structures constituted dilatations/diverticula of the one of a pair of twins from a polled Toggenburg dam and polled 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 di- 1Research Service (151), Department of Veterans Affairs, Edward Hines Jr. VA Hospital, agnoses of bilateral cryptorchidism, segmental urethral 2 Hines, Illinois 60141; Department of Pathology, Loyola University Medical Center, Maywood, hypoplasia, and urethral diverticula. Illinois 60153; 3House-Call Veterinary Service, Lockport, Illinois 60441 *Corresponding author: William W. King, DVM, PhD, Diplomate, ACLAM, Office of The preputial ambiguity strongly suggested a genetic condi- Research Services, School of Medicine, University of Louisville, Louisville, Kentucky 40292 tion involving reproductive development. In neonatal ruminants,

Volume 41, No. 5 / September 2002 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 Congenital abnormalities. Developmental anomalies may re- sult from both genotypic and environmental influences, or a combination of both, with an interruption of the “temporal-spa- tial 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 ori- fice (e.g. , with exstrophy or eversion of the bladder), urethral aplasia and agenesis, ectopic urethra, ure- thral duplication, and urethrorectal or urethrovaginal fistula (11, 12). In goats, inherited anomalies associated with the distal uri- nary tract and/or reproductive tract include bipartite scrotum Figure 1. External appearance of anomalies. Note the two ventral mid- and cryptorchidism in Angora goats, testicular hypoplasia, sperm line cyst-like masses and the ambiguous prepuce. granulomas, and intersex (7). Reports of urethral diverticula in neonatal goats are uncom- mon. 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 ) 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 inconsisten- Figure 2. Location of cryptorchid (A) in subcutaneous inguinal cies in goats is “intersex” (7). This condition results from sex canal (B, vaginal ring). chromosomal abnormalities and has been defined as “ambigu- ity in the structure of the gonads, reproductive tract, or external the distal aspect of the penis, including the urethral process in genitals” (14). The disorder is strongly associated with the polled goats, is adhered to the prepuce (9). However, the testicles nor- condition in goat breeds of western European descent (Saanen, mally are descended at birth into a well-developed scrotum (7). Alpine, and Toggenburg) and is less commonly found with In this case, only a rudimentary penile structure was identifi- freemartinism (15, 16). able, the prepuce was located in an intermediate position, and, There have been several descriptive surveys of the intersex unlike in the surviving twin male, no scrotum was evident. In condition in dairy goats. Hamerton and colleagues examined light of these findings, the known parentage, and the similari- 35 cases of polled intersexuality and found that the vast majority ties with other clinical reports, the presumptive etiology for the (97%) of cases were genetically female (60,XX) with mixed male anomalies seen in this case is the intersex condition seen in polled phenotype and that there was a low percentage (3%) of breeds. Limitations in the tissues available for additional diag- freemartins (60,XX/60,XY) (15). In 1943, Eaton described 29 nostic evaluation made definitive differentiation from cases of “,” classifying each into four freemartinism impossible. “female-predominant” and four “male-predominant” categories

40 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science 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 open- Figure 4. Breeding schemes associated with the intersex condition. Note ing. Other findings reported to occur in conjunction with the that a homozygous polled doe (PP) is infertile and therefore not shown. intersex condition include cryptorchidism, in which the testes (Modified from Smith and Sherman, 1994.) typically are retained within the abdomen but may be partially or fully descended (16). chimerism (25, 26). Unfortunately, appropriate tissues were not A more recent report involving an 18-day-old intersex French- archived from the described goat to perform these confirma- Alpine/Nubian goat had remarkable similarities to the case tory steps. However, the prevalence of placental fusion is much described in the present report (18). This kid presented with a lower in goats than in , making freemartinism much less ventral midline urethral dilatation associated with a hypoplastic common in goats (7). distal urethra at the prepuce. The urethra was found to be dis- Reproductive management. Institutions and animal breeders tinctly separate from a rudimentary penis; surgical correction producing goats may find the congenital lack of horns a desir- and urethrostomy was performed. During an exploratory abdomi- able characteristic with practical and humane advantages over nal surgery for gonadectomy, testicles were located adjacent to surgical dehorning or disbudding. However, there are several each . Blood analysis revealed that the goat was problems associated with the polled condition. An understand- genotypically female (60,XX). ing of the mechanisms of transmission of PIS therefore is Genetic anomalies. Gender development results from com- extremely important when designing a production program. plex interactions of sex- and autosomal-linked . The SRY Figure 4 illustrates the breeding crosses that may result in inter- (sex-determining region Y) directs mammalian embryos sex offspring. to develop testes, which subsequently support the male pheno- The intersex condition in European goats can be avoided by type by producing Mullerian inhibiting proteins and ensuring that one parent was born with horns (genotypically (19, 20). This cascade of genetic events is not simple, however, horned), that is, homozygous recessive for the polled gene (p). as is illustrated in the intersex condition seen in polled goats. This precaution will prevent any female offspring from express- The interaction between hornlessness and intersex in geneti- ing the intersex associated with the homozygous dominant polled cally female (XX) goats, first suggested by Asdell in 1944 (21), gene (P) (7). Note that a central whorl of hair on the dorsal has been dubbed the Polled Intersex Syndrome (PIS) and is cranium is present in polled goats; two whorls overlying the area thought to be caused by an interference with the development of horn growth is present in disbudded kids (7). of female characteristics by aberrant expression of male-promot- In programs designed to increase the number of polled off- ing gene products (7). spring, the goals of increasing and decreasing the Phenotypically, the mutation (P), which is either a single potential for often conflict. To maximize reproduc- pleiotropic gene or two intimately linked genes, expresses tive output, breeding horned bucks (pp) to fertile (therefore polledness in an autosomal dominant fashion and results in heterozygous, Pp) polled does, which often produce more off- sex-reversal in females as an autosomal recessive trait (22). The spring including twins and triplets (16), will result in only 50% mutation partially simulates the , encouraging of the offspring being polled but with no possibility of PIS fe- the development of testicular tissue by generating H-Y antigen males. Although there are disadvantages in using homozygous (16, 23, 24). Interestingly, the PIS XX intersex is SRY-negative, polled bucks (PP), which frequently develop sperm granulomas a fact that supports theories of autosomal gene involvement in resulting in obstruction of the head of the epididymis and tes- male gender development independent the Y-chromosome or ticular hypoplasia (7, 16), the cross of such a sire with a of X-Y or autosomal-Y translocation of SRY genes (25, 26). The genotypically horned doe (pp) results in 100% heterozygous mutation causing PIS recently has been identified as an 11.7- polled offspring, also with no potential for producing polled- kb deletion on chromosome 1 (20, 27, 28). Females that are associated intersexes. Crossing a polled buck (regardless of homozygous for the polled gene (PP) will become infertile in- genotype) with a doe born horned (pp) will result in a 6/8 tersex (7); fertile polled females therefore must be heterozygous (75.0%) probability of polled offspring with no possibility of PIS for the polled gene (Pp) (16). females. Breeding a polled buck (again, regardless of genotype) Intersex secondary to freemartinism is another possibility in with a polled doe (Pp) will result in a 7/8 (87.5%) probability of the described case. The pathogenesis of this condition involves polled offspring but a concurrent 3/8 (37.5%) chance of inter- the sharing of cells and hormones between a male and female sex in female kids. fetus following placental fusion and anastomosis, resulting in Alternatively, breeding schemes can be designed to specifi- chimerism in the female twin (16). Karyotype analysis gener- cally produce PIS animals for use as models of the human ally is used to differentiate polled intersex (genetically female, XX-male intersex condition. In humans, conditions leading to XX) from freemartinism (chromosomal chimerism, XX/XY). intersex may occur at any time during intrauterine development This assay can be performed on lymphocytes from whole blood and may lead to a spectrum of consequences ranging from overt or cultured renal or pericardial fibroblasts (10). Several authors anatomical and physiological anomalies to more elusive abnor- have described the use of polymerase chain reaction (PCR) tech- malities not appreciated until sexual maturity (31). Although niques for determining sex in goats (29, 30). Examination of considerable work has focused on the appropriate timing of treat- fresh tissue with either PCR-based assays or Southern blot for ment, intersexuality continues to result in a significant incidence the presence of the Y-chromosome-associated Zfy and Sry re- in gender identity disorders (32). The presentation of such in- gions can differentiate polled intersex from freemartin fants produces a two-fold crisis: 1) challenging medical and

Volume 41, No. 5 / September 2002 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science 41 surgical interventions, and 2) extensive sociological and psycho- 13. Gahlot, T. K., A. K. Ranka, D. S. Chouhan, et al. 1982. Congenital logical adjustment (33, 34). Of particular interest, it has been urethral deverticulum [sic] in male goat (Capra hircus)—surgical recently suggested that the PIS gene is homologous to the locus management. Indian J. Vet. Surg. 3:95-97. linked to blepharophimosis ptosis epicanthus inversus syndrome 14. Noden, D. M. and A. de Lahunta. 1985. The embryology of domes- tic animals. Developmental mechanisms and malformations. (BPES), a condition associated with ovarian dysfunction and Williams & Wilkins, Baltimore. epidermal thickening of the eyelid in affected humans (27, 35). 15. Hamerton, J. L., J. M. Dickson, C. E. Pollard, et al. 1969. 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42 CONTEMPORARY TOPICS © 2002 by the American Association for Laboratory Animal Science Volume 41, No. 5 / September 2002