History
A 2-day-old 2.27-kg (5.0-lb) male crossbred goat kid was admitted to the J. T. Vaughan Large Animal Teaching Hospital at Auburn University for evaluation of abnormalities of the external genitalia. A female twin was also admitted concurrently but had no visible abnormalities. The kids were offspring resulting from a mating between a polled pygmy crossbred doe and a full-sibling polled buck (same sire and dam as the doe). The owner reported that both kids were suckling normally.
No further examinations were performed on the female kid. The male kid closely resembled a phenotypic male because of the anogenital distance and presence of external male features, including a penis, preputial sheath, and prepuce; however, the penis was hypoplastic, and there was no evidence of a scrotum. The kid was bilaterally cryptorchid, but both presumptive gonads were palpable (1 in each inguinal region). A thin-walled, fluid-filled structure (4 × 8 × 2 cm) extended from the midportion of the preputial sheath to the perineum (Figure 1).
Photograph of the perineal region of a 2-day-old phenotypically male goat kid with a cyst-like structure. The kid was born cotwin to a female; the offspring were the result of a mating between a polled pygmy crossbred doe and a full-sibling polled buck (same sire and dam as the doe). The tail of the goat is at the bottom of the image.
Citation: Journal of the American Veterinary Medical Association 248, 10; 10.2460/javma.248.10.1121
The exposed surface of the fluid-filled structure was smooth, translucent, hairless, and well vascularized. The hypoplastic distal portion of the penis was palpable through the preputial sheath between the distal end of the fluid-filled dilatation and the external preputial orifice, but the penis could not be exteriorized. Gentle manual pressure applied to the cystic structure resulted in the expulsion of drops of colorless fluid (which had the odor of urine) from the external preputial orifice. The expulsed drops were collected and submitted for analysis. Laboratory results were consistent with those for a urine sample. An attempt to pass a urethral catheter through the external preputial orifice and into the cyst was unsuccessful. Results of a CBC and serum biochemical analysis were within reference limits.
Initial treatment consisted of frequent application of an antimicrobial ointment and humectants to protect the urethral cyst from desiccation. During the next several days, the exposed surface of the urethral cyst developed an inflamed appearance and became friable, and the kid had difficulty completely emptying the bladder during urination. Thus, it was elected that a urethrostomy be performed.
Prior to surgery, fluoroscopic evaluation with contrast medium was attempted to define the anomaly. The kid was sedated with xylazine hydrochloride (0.1 mg/kg [0.045 mg/lb], IM), and an attempt was made to insert a 3.5F × 13-cm tom cat urinary catheter into the distal portion of the urethra so that retrograde urethrography could be performed. The catheter could be advanced only 1 mm into the preputial orifice, and infusion of the contrast medium met with great resistance, which resulted in outflow around the catheter.
Immediately after unsuccessful urethrography was completed, the kid was intubated with a tracheal tube. Anesthesia was maintained by administration of isoflurane. The urethrostomy site was aseptically prepared. The urethral cyst was incised along the midline on its long axis, and the most pendulous portion of the urethral tissue was excised. After the lumen of the cyst was incised, a urinary catheter could easily be passed into the proximal portion of the penile urethra, which confirmed that the urethra was the origin of the cyst. Excess urethral tissue was excised, and the cut edges of the urethra were sutured to the adjacent skin on both sides, which increased the urethral opening to allow urine drainage. Urine passed freely from the urethrostomy site, and the kid recovered from anesthesia without complications. Ceftiofur sodium (4.4 mg/kg [2 mg/lb], SC, q 24 h for 5 days) and flunixin meglumine (1.1 mg/kg [0.5 mg/lb], IV, q 12 h for 3 days) were administered after surgery. In addition, an antimicrobial ointment was applied topically to the surgery site every 12 hours for 14 days.
Question
What is the most likely cause of the developmental anomalies of sexual differentiation in this goat?
Answer
In a kid that was the offspring resulting from a mating in which both the dam and sire were polled, the most likely explanation for ambiguities in the external genitalia and bilateral cryptorchidism is polled intersex syndrome (PIS) of goats.
Results
A blood sample was obtained from the kid and submitted to the Molecular Cytogenetic and Genomics Laboratory in the Department of Veterinary Integrative Biosciences at Texas A&M University for karyotypic analysis. Results of examination of cells indicated a female karyotype (60, XX; Figure 2). Results of a quantitative PCR assay for the presence of the sex-determining region of the Y chromosone (SRY) were negative, which indicated that the kid was genetically female (ie, XX chromosome complement and no SRY gene).
Photograph of the karyotype (A) and an electrophoretic gel for SRY determination (B) of the phenotypically male goat kid in Figure 1. Lanes in panel B are as follows: M = 1,000-bp ladder, 1 = sample obtained from the phenotypically male goat kid, 2 = sample obtained from a genotypically normal control male goat, and 3 = sample obtained from a genotypically normal control female goat. In panel A, notice that there is a phenotypically normal female chromosome complement (60, XX). In panel B, the gel reveals absence of the SRY (arrow) in the goat kid of Figure 1.
Citation: Journal of the American Veterinary Medical Association 248, 10; 10.2460/javma.248.10.1121
Thirty days after urethrostomy and marsupialization of the diverticulum were performed, the kid was castrated. The kid was again anesthetized with isoflurane and positioned in dorsal recumbency. The gonads could easily be palpated beneath the dermis overlying the inguinal rings. An incision was made through the skin and subcutaneous tissues overlying each gonad; each gonad was then accessed by blunt dissection. The gonads were removed by use of a closed castration technique. Both gonads appeared to be testes on gross visual inspection; they were fixed in formalin and submitted for histologic evaluation. The kid again recovered from surgery without complications.
Histologic examination was performed on the formalin-fixed gonads. Multiple 4-µm-thick tissue sections were cut from each gonad. Sections were stained with H&E stain and microscopically examined. Histologic evaluation revealed that the gonads were typical of a prepubertal male, with histologically normal testicular and epididymal tissues (Figure 3). Well-developed seminiferous tubules were devoid of developing spermatogonia. No structures resembling ovarian tissue were detected during examination of any of the stained sections.
Photomicrograph of a section of testicular tissue from the goat kid of Figure 1. Notice the well-defined seminiferous tubules with no active spermatogenesis. H&E stain; bar = 100 μm.
Citation: Journal of the American Veterinary Medical Association 248, 10; 10.2460/javma.248.10.1121
Further information on the origin of the disorder of sexual differentiation in this kid was obtained. A sample of DNA from the kid was submitted to the Institut National de La Recherche Agronomique France and tested for the PIS-associated deletion on chromosome 1. Presence or absence of the PIS region of interest was determined by use of a quantitative PCR assay with primers for the deleted region. Genotype for PIS was determined by comparing the PIS-to-actin B ratio of the kid with the ratio for animals of known PIS genotypes (PIS control animals). Results indicated that the kid was homozygous for the PIS deletion (PIS –/–; Figure 4).
Results for a quantitative PCR assay to determine the PIS status of the phenotypically male goat kid of Figure 1. The PIS-to-actin B ratio for the various PIS genotypes is as follows: homozygous for PIS, PIS +/+; heterozygous for PIS, PIS +/–; and homozygous for the PIS deletion, PIS –/–. Notice that the goat kid is homozygous for the deletion (PIS –/–).
Citation: Journal of the American Veterinary Medical Association 248, 10; 10.2460/javma.248.10.1121
Discussion
The kid of the present report had readily identifiable disorders of sexual development, including cryptorchidism, incomplete development of the penis, and a urethral diverticulum leading to a cyst-like structure on the ventral aspect of the prepuce. A presumptive diagnosis of PIS can be made on the basis of the medical history and physical examination findings, but other disorders of sexual development cannot be excluded without further testing. Other differential diagnoses for disorders of sexual development in this kid, including aneuploidy, chimerism, and female pseudohermaphroditism, were less likely on the basis of the clinical findings and history.
Polled intersex syndrome of goats is the result of an 11.7-kb deletion of the polled intersex transcribing region of chromosome 1.1 The PIS deletion results in disruption of transcriptional regulation for the factor Forkhead box L2 (FOXL2) and is associated with lack of development of horn buds in animals that are heterozygous or homozygous for the deletion.2 The PIS-associated disorders of sexual development are seen in chromosomally female goats (60, XX) that are homozygous for the 11.7-kb PIS deletion on chromosome 1, which is associated with the polled condition. In females homozygous for the 11.7-kb PIS deletion on chromosome 1, dysregulation of gonadal development is followed by formation of testes or ovotestes rather than ovaries.2,3 Polled intersex goats are examples of XX sex reversal, which is characterized by a 60 XX female chromosomal complement and a male gonadal phenotype. Molecular testing is required to determine the PIS deletion.
Presumptive identification of PIS as the cause of abnormal sexual development requires karyotypic analysis to confirm a 60 XX female chromosome complement, testing to determine concurrent absence of the SRY, and histologic confirmation of testicular tissue in the gonads. Definitive diagnosis of PIS requires molecular testing to determine that the affected animal is homozygous for the PIS deletion mutation (PIS –/–) that is responsible for abnormal sexual development.
Reports of disorders of sexual development related to the polled gene in goats can be traced to the end of the 19th century. Recognition of the strong association for disorders of sexual differentiation in goats homozygous for genes controlling the polled condition led to a long-standing recommendation to include at least 1 horned parent (sire or dam) when mating goats.
The PIS genotype associates the absence of horns with female-to-male sex reversal in biallelic affected XX animals.2 The deleted 11.7-kb DNA fragment encompasses no coding regions but exerts transcriptional regulatory effects on 3 genes: polled intersex syndrome regulated transcript 1 (PISRT1), FOXL2, and promotor FOXL2 inverse complementary (PFOXic).2–4 The expression of these genes in gonadal tissues is dependent on the PIS genotype. In an XX gonad, all 3 genes and long transcripts are expressed from the beginning of ovarian formation until adulthood in animals homozygous for PIS (PIS +/+) and heterozygous for PIS (PIS +/–), whereas expression is disrupted in animals homozygous for the PIS deletion (PIS –/–).2
Important steps in gonadal differentiation are controlled by the sex-chromosome content of an animal. In goats, the genital ridges arise from the mesonephros at approximately 23 to 25 days after coitum; gonadal FOXL2 transcription typically is initiated in a subpopulation of somatic cells located in the central medulla of an XX gonad between 34 and 36 days after coitum,2 with the first signs of ovarian differentiation detectable by 36 days after coitum.2,3 These developmental stages correspond to the gonadal switch during which male sex determination is initiated by an increase in sex-determining region Y–box 9 (SOX9) expression and guides differentiation to testis formation in an XY gonad. In 60 XX gonads and gonads heterozygous for PIS (PIS +/–), SOX9 expression is inhibited by FOXL2 and perhaps by PISRT1 expression. Biallelic PIS mutation in an XX animal results in absence of the typical FOXL2 and PISRT1 transcription in the developing gonads beginning at 34 to 36 days after coitum because SOX9 expression is not inhibited as a result of failure of expression of PISRT1 and FOXL2 and because there is differentiation of the somatic cells of the gonads into testicular cells, with a delay of 4 to 5 days in development.1,3,4 This explains the histologic findings for the gonads of the goat of the present report in which there were seminiferous tubules; however, the seminiferous tubules were devoid of developing spermatogonia.
Although congenital anomalies of the urinary system develop rarely in ruminants, with hypospadias and urethral diverticulum being reported sporadically, ambiguous genitalia is a common feature in PIS –/– goats. Multiple congenital anomalies, including urethral diverticula, urethral stenosis, testicular and penile hypoplasia, and cryptorchidism, are often detected in the same animal. It has been reported5 that 13% of polled intersex goats with male genitalia had hypospadias. Hypospadias develops as a result of imperfect closure or complete lack of fusion of the urethral grooves.
The urethral dilatation associated with a hypoplastic distal portion of the urethra located on the ventral aspect of the sheath at an area distal to where the sigmoid flexure would develop in the goat described here represented an anomalous closure of the ventral portion of the penis. This defect has been reported in other goat kids with PIS.6 It can be hypothesized that this failure of closure is related to a delay in androgen hormonal signaling because there is no gonadal differentiation at the appropriate number of days after coitum in sex-reversed goats.
Affected goat kids often have scrotal anomalies, including a bipartite scrotum or complete absence of the scrotum, as was the situation in the goat of the present report. These animals may have multiple anomalies; however, these animals can be functional and appear to have a lifespan that is similar to that of their contemporaries.
Outcome
The castrated goat was kept as a pet. All surgical sites healed without complication. One year after castration, the goat had no other medical issues and continued to urinate through the urethrostomy without difficulty.
Acknowledgments
The authors thank the Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University for providing the image of the karyotype in Figure 2; Dr. Joseph Newton for providing Figure 3; and Dr. Eric Pailhoux and Marjolaine André for assistance with genotyping.
References
1. Pailhoux E, Vigier B, Chaffaux S, et al. A 11.7-kb deletion triggers intersexuality and polledness in goats. Nat Genet 2001; 29: 453–458.
2. Boulanger L, Pannetier M, Gall L, et al. FOXL2 is a female sex-determining gene in the goat. Curr Biol 2014; 24: 404–408.
3. Pannetier M, Elzaiat M, Thepot D, et al. Telling the story of XX sex reversal in the goat: highlighting the sex-crossroad in domestic mammals. Sex Dev 2012; 6: 33–45.
4. Pailhoux E, Vigier B, Schibler L, et al. Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation. Genet Sel Evol 2005; 37 (suppl 1): S55–S64.
5. Eaton O. An anatomical study of hermaphrodism in goats. Am J Vet Res 1943; 4: 333–343.
6. King WW, Young ME, Fox ME. Multiple congenital genitourinary anomalies in a polled goat. Contemp Top Lab Anim Sci 2002; 41: 39–42.
