J Toxicol Pathol 2007; 20: 111–115

Case Report Bilateral Ovotestes in a Female Beagle Dog

Kinji Kobayashi1, Toshihisa Fujiwara1, Tamiko Adachi1, Masatoshi Asahina1, Yoshifumi Sasaki1, Aoi Matsuda1, Tomonari Nishimura1, Toshihide Inui1, and Kazuyuki Kitamura1

1Drug Safety Research Laboratories, TANABE Seiyaku Co., Ltd., 16–89 Kashima 3-chome, Yodogawa-ku, Osaka 532–8505, Japan

Abstract: We report a case of bilateral ovotestes in a female beagle dog. This dog was used in a 4-week repeated- dose toxicity study and sacrificed by exsanguination at 6 months of age. Clinical observation, hematological examination, blood chemistry analysis, urinalysis and autopsy did not reveal any abnormal or drug-induced effects. Microscopically, seminiferous-like tissue was observed in the medulla of the bilateral ovaries. The morphological and immunohistochemical characteristics of Sertoli-like cells lining the seminiferous-like tubules corresponded to those of Sertoli cells in the testis. Myoid-like cells exhibiting positive reactions for alpha smooth muscle actin surrounded the seminiferous-like tubules. Therefore, the dog was regarded as a case of true hermaphroditism with bilateral ovotestes. PCR using DNA extracted from paraffin-embedded sections of the testicular parts of the ovotestis, uteri and spleen showed that the organogenesis of ovotestes in this case was not associated with the sex-determining region Y gene. (J Toxicol Pathol 2007; 20: 111–115) Key words: ovotestes, true hermaphroditism, SRY, beagle

Ovotestis refers to a gonad with histology consistent (1:200, DAKO Japan Co., Ltd., Kyoto, Japan), anti-inhibin- with both ovarian follicles and testicular tubular elements. alpha (1:50, DAKO), anti-alpha smooth muscle actin (SMA) Such gonads are frequently found in animals with true (used as supplied, DAKO) and anti-proliferation cell nuclear hermaphroditism1, and canine ovotestes have been associated antigen (PCNA) (1:200, DAKO) antibodies. The tissue with XX sex reversal and true hermaphroditism2–6. Here, we sections were boiled in Instant antigen activator agent H report bilateral ovotestes observed in a female beagle dog (neutral) (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan), with an otherwise normal appearance. The dog was incubated with a 0.3% (v/v) methanol solution of hydrogen purchased from Hongo Farm (Kitayama Labes Co., Ltd., peroxide for quenching of endogenous peroxidase, and Yamaguchi, Japan) and housed at Tanabe Seiyaku Co. Ltd. immersed in normal goat serum (used as supplied, Vector (Osaka, Japan). The animal was treated with a particular Laboratories, Inc., Burlingame, CA, USA) to prevent non- chemical in a 4-week repeated-dose toxicity study approved specific reactions. The antibodies were applied to the tissue by the Animal Ethics Committee of Tanabe Seiyaku Co., sections overnight at 4°C and were subsequently detected Ltd. Clinical observation, hematological examination, blood using Histofine® Simple Stain MAX PO (MULTI) (Nichirei chemistry analysis and urinalysis revealed no abnormalities Co., Ltd., Tokyo, Japan) and/or Histofine® Simple Stain or drug-induced effects. At 6 months of age, the animal was DAB solution (Nichirei). The seminiferous-like tubules in sacrificed by exsanguination under thiopental sodium the ovotestes were compared immunohistochemically with anesthesia and an autopsy was conducted. The ovaries were seminiferous tubules in the testis of a male beagle, granulosa fixed in 10% phosphate-buffered formalin solution and cell cords in the ovary of a female beagle (both of these embedded in paraffin. Tissue sections were stained with animals were in the control group in the same toxicity study) hematoxylin and eosin. and hyperplasia of the granulosa cell cords, which is Immunohistochemistry was performed with primary histologically similar to our case, in the ovary of a female antibodies, including mouse monoclonal anti-vimentin mongrel dog (aged 12 years old). DNA samples were prepared from paraffin- Received: 21 February 2007, Accepted: 18 April 2007 embedded sections of the testicular regions (medulla) in Mailing address: Kinji Kobayashi, Drug Safety Research Laboratories, the ovotestis, of the medulla in the ovaries, uteri and TANABE Seiyaku Co., Ltd., 16–89 Kashima 3-chome, Yodogawa-ku, spleens of the case animal and control animals, and of the Osaka 532–8505, Japan testis of the control animal using DNA Isolator PS-Rapid TEL: 81-6-6300-2958 FAX: 81-6-6300-2696 Reagent (Wako Pure Chemical Industries Ltd., Osaka, E-mail: [email protected] 112 Canine Ovotestes

Fig. 1. Histopathology of the ovotestes in a beagle dog. A mass of tubular-like tissue was found in the medulla of the ovary (a). The tubular tissue was similar in structure to the seminiferous tubules and lined by spindle cells (Sertoli-like cells) with nuclei located in basal area (b). HE staining; Bar = 250 µm (a); Bar = 50 µm (b).

Fig. 2. Immunohistopathology for alpha smooth muscle actin (SMA) in the seminiferous-like tubules of the canine ovotestes. The cells surrounding the seminiferous-like tubules in the ovotestis (a; arrowheads) and the testis (c) were positive for SMA, whereas positive reactions for SMA were not observed around the granulosa cell cords in the ovary of a control animal (b). Bar = 30 µm.

Japan). The primer (-1) and nested primer (-2) sequences TAC-3'; HPRT forward primer-2, 5'-TGG CTA TAA ACC for amplification of the canine sex-determining region Y TGA CTG TAA GTG-3'; and HPRT reverse primer-2, 5'- (SRY) gene on the Y-chromosome and the hypoxanthine CGT GCC TTC TCT ACA AAT ACT CTC-3'. The partial phosphoribosyltransferase (HPRT) gene on the X- sequences of the canine HPRT and SRY genes were chromosome were as follows: SRY forward primer-1, 5'- amplified for 32 cycles in a thermal cycler (PTC-200 DNA GAC GAC CCA TGA ACG CAT TCT TGG-3'; SRY Engine™, MJ Research, Watertown, MS) using the reverse primer-1, 5'-CTC GAA GAA TGG CCA TTT TTC following conditions: initial denaturation at 94°C for 2 min; GGC-3'; SRY forward primer-2, 5'-GAA CGC ATT CTT 10 cycles at 94°C for 30 sec, at 58°C (HPRT)/62°C (SRY) GGT GTG GTC TC-3'; SRY reverse primer-2, 5'-GGC CAT for 30 sec, and at 72°C for 45 sec; 20 cycles at 94°C for 30 TTT TCG GCT TCT GTA AG-3'; HPRT forward primer-1, sec, at 56°C (HPRT)/60°C (SRY) for 30 sec, and at 72°C for 5'-CCC TCG AAG TGT TGG CTA TAA ACC-3'; HPRT 45 sec; and a final extension step at 72°C for 1 min. The reverse primer-1, 5'-TGC TTA CAA ACG TGC CTT CTC expected sizes of PCR products for the SRY and HPRT genes Kobayashi, Fujiwara, Adachi et al. 113 were 132 base pairs (bp) and 94 bp, respectively. DNA sequencing analysis of the amplicons from nested PCRs obtained for the testis of the control animal was performed at Dragon Genomics Center (Takara Bio, Inc., Shiga, Japan) and showed that the products were derived from the SRY (EMBL: AF107021) and HPRT (EMBL: AY283373) genes. Macroscopically, there were no abnormalities of the external or internal genitalia, including the ovaries, in this case, although XX true hermaphrodite dogs typically show ambiguous (partially masculinized) external genitalia2,3,5,6. The weights of the ovaries were similar to those of female beagles in the control group. Microscopically, a mass of tubular-like tissue was noted in the medulla of the bilateral ovaries (Fig. 1a). The tubular tissue was similar to atrophied seminiferous tubules and was lined by spindle cells (Sertoli- like cells) with nuclei located in the basal area (Fig. 1b). Fig. 3. PCR results for canine SRY and HPRT genes using DNA Spermatogenic germ cells were not observed within the extracted from paraffin-embedded sections of the testicular seminiferous-like tubules. Round or polygonal cells regions (medulla) in the ovotestis, the medulla in the ovary, (Leydig-like cells) with granular eosinophilic cytoplasm and and the testis. Lane a: 100 base-pair (bp) molecular weight marker; lanes b and e: testis; lanes c and f: ovotestis; lanes d small round nuclei were scattered between the seminiferous- and g: ovary. The expected sizes of PCR products for the SRY like tubules. In addition, there were fewer oocytes and HPRT genes are 132 bp and 94 bp, respectively. (primordial follicles) in the cortex of the ovaries in the case animal compared to the control animals, but primary follicles, secondary follicles, Graafian follicles, corpora lutea, and germinal epithelia did not have any abnormalities. diagnosis of the lesion as bilateral ovotestes. Since true Immunohistochemically, the Sertoli-like cells were hermaphrodites have both ovarian and testicular tissues, negative for PCNA and positive for vimentin. These cells either in the form of a combined gonad (ovotestis) or as showed varying degree of positive reactions for inhibin- separate organs, this case was regarded as an example of true alpha. The cells lining the granulosa cell cords in the ovary hermaphroditism. of the control female beagle were positive for PCNA and Canine ovotestes have been reported in cases of XX sex vimentin, and strongly positive for inhibin-alpha; and the reversal and true hermaphrodites2–6. Peripheral Sertoli cells in the testis of the control male beagle were analysis was not performed in the current case, but PCR negative for PCNA, positive for vimentin, and weakly primers targeting the SRY gene were used to judge whether positive for inhibin-alpha. The cells lining the hyperplastic the animal had a Y chromosome. Since the SRY gene was granulosa cell cords in the ovary of the female mongrel dog not amplified in PCR using DNA samples isolated from were positive for PCNA and vimentin, and mostly negative paraffin-embedded sections of the spleen, it was judged that for inhibin-alpha. Cells exhibiting positive reactions for the case animal did not have a Y chromosome. alpha smooth muscle actin were seen around the Phenotypic, gonadal, and molecular studies have led to seminiferous-like tubules in the case (Fig. 2a) and the several hypotheses for the developmental mechanism of seminiferous tubules in the testis of the control male dog ovotestes, including genetic chimerism due to double (Fig. 2c), whereas positive reactions for alpha smooth fertilization or an exchange of cells between dizygous twins muscle actin were not observed around the granulosa cell of different sex7–9; mitotic non-disjunction on the Y cords in the ovary of the control female dog (Fig. 2b) or in chromosome at the first two cleavage divisions10,11; the hyperplastic granulosa cell cords in the female mongrel translocation Y-chromosomal sequences, including the SRY dog. In addition, the Leydig-like cells between the gene, onto an X chromosome12; of a gene on the X seminiferous-like tubules were negative for PCNA and chromosome or alternatively on an autosome that allows positive for vimentin. The Leydig-like cells in the testis of testis determination without the SRY gene13; and hidden the control male dog were clearly positive for inhibin-alpha, mosaicism with Y-bearing cells in the gonads14,15. We but these cells showed variable affinity for anti-inhibin- evaluated SRY hidden gonadal mosaicism in this case by alpha antibody in the case animal. The Sertoli-like cells had SRY gene amplification using DNA samples isolated from morphological and immunohistochemical characteristics paraffin-embedded sections. As a result, although a HPRT corresponding to those of actual Sertoli cells, and myoid-like gene as positive control was amplified, a hidden SRY gene cells exhibiting positive reactions for alpha smooth muscle was not found in the ovotestes or (Fig. 3). Therefore, actin surrounded some of the seminiferous-like tubules. The we concluded that the SRY gene was not associated with the results identify the tubular structures in the medulla of development of ovotestes in this case. ovaries as seminiferous tubules, rather than normal or Expression of SRY might initiate testis induction by up- hyperplastic granulosa cell cords, allowing histopathological regulating SRY-related high-mobility group box 9 (Sox9) 114 Canine Ovotestes expression16. The timing of SRY and SOX9 expression is 5. Sommer MM and Meyers-Wallen VN. XX true consistent with a role in testis determination in dogs, as well hermaphroditism in a dog. J Am Vet Med Assoc. 198: 435– as in humans, sheep and pigs17. However, SOX9 is up- 438. 1991. regulated by loss of WNT4 in XX gonads, even if SRY is 6. Fitzgerald AL and Murphy DA. Bilateral ovotestes in an absent18, and loss of WNT4 gives rise to masculinization of , mixed breed dog. Lab Anim Sci. 40: 647–649. 1990. the XX gonad19. There were fewer oocytes in the cortices of 7. Schoenle E, Schmid W, Schinzel A, Mahler M, Ritter M, the ovotestes in the case animal, compared with the control. Schenker T, Metaxas M, Froesch P, and Froesch ER. 46,XX/ Since it is thought that WNT4 acts through follistatin to 46,XY chimerism in a phenotypically normal man. Hum 20 maintain survival in the ovary , WNT4 deficiency Genet. 64: 86–89. 1983. may induce a decrease in oocytes. It has also been reported 8. Verp MS, Harrison HH, Ober C, Oliveri D, Amarose AP, that exogenous administration to a pregnant bitch Lindgren V, and Talerman A. Chimerism as the etiology of a caused true hermaphroditism with the presence of bilateral 46,XX/46,XY fertile true hermaphrodite. Fertil Steril. 57: ovotestis and remnants of the Wolffian duct system in all 346–349. 1992. female pups21. Because WNT4 overexpression inhibits 9. Uehara S, Nata M, Nagae M, Sagisaka K, Okamura K, and testosterone synthesis by repressing / Yajima A. Molecular biologic analyses of tetragametic beta-catenin synergy22, loss of WNT4 in a female may chimerism in a true hermaphrodite with 46,XX/46,XY. Fertil Steril. 63: 189–192. 1995. enhance testosterone synthesis. Aromatase is a catabolizing 10. Whitten WK, Cater SC, and Beamer WG. Sex reversing enzyme that controls the androgen/estrogen ratio by non-disjunction of the Y chromosome produces 23 irreversible conversion of testosterone into estradiol , and exceptionally low sex ratio (% males) and hermaphrodites in aromatase inhibitors administered before sexual the progeny of male BALB/cBm mice: the roles of the differentiation of the gonads can induce transdifferentiation maternal genotype and the Y chromosome. Reprod Fertil from ovarian medullary cords into testicular cords/tubes, Dev. 3: 255–265. 1991. causing sex reversal in fish23, turtles24 and chickens25. 11. Bean CJ, Hassold TJ, Judis L, and Hunt PA. Fertilization in Therefore, exposure to excess testosterone in the gonads vitro increases non-disjunction during early cleavage before sexual differentiation may be a cause of ovotestis. divisions in a mouse model system. Hum Reprod. 17: 2362– The mutation causing canine SRY-negative XX sex 2367. 2002. reversal does not seem to be located in regions containing 12. Kusz K, Kotecki M, Wojda A, Szarras-Czapnik M, Latos- Bielenska A, Warenik-Szymankiewicz A, Ruszczynska- several candidate genes (WT1, DMRT1, GATA4, FOG2, 26–29 Wolska A, and Jaruzelska J. Incomplete masculinisation of LHX1, SF1, SOX9 or Lhx9) . Mutation of the WNT4 gene XX subjects carrying the SRY gene on an inactive X is a further possible candidate as a cause of canine SRY- chromosome. J Med Genet. 36: 452–456. 1999. negative XX sex reversal, and it remains to be seen whether 13. Kojima Y, Hayashi Y, Asai N, Maruyama T, Sasaki S, and loss of WNT4 occurred in the current case. Quantitation of Kohri K. Detection of the sex-determining region of the Y plasma sex hormones and aromatase activity in the gonad chromosome in 46,XX true hermaphroditism. Urol Int. 60: may also shed light on the cause of canine ovotestes. 235–238. 1998. 14. Inoue H, Nomura M, Yamase T, Ichino I, Goto K, Ikuyama Acknowledgements: The authors would like to thank Dr. S, Takayanagi R, and Nawata H. A rare case of 46,XX true Mitsuru Kuwamura of Osaka Prefectural University for his hermaphroditism with hidden mosaicism with sex- kind gift of paraffin-embedded sections of the ovary of a determining region Y chromosome-bearing cells in the gonads. Intern Med. 37: 467–471. 1998. female mongrel dog. We are also grateful to all attendees of 15. Queipo G, Zenteno JC, Pena R, Nieto K, Radillo A, the Kansai Conference on Toxicologic Pathology for the Dorantes LM, Erana L, Lieberman E, Soderlund E, Jimenez histopathological review. AL, Ramon G, and Kofman-Alfaro S. Molecular analysis in true hermaphroditism: demonstration of low-level hidden References mosaicism for Y-derived sequences in 46,XX cases. Hum Genet. 111: 278–283. 2002. 1. Jones TM, Hunt RD, and King NW. In: Ambiguous sexual 16. Kobayashi A, Chang H, Chaboissier MC, Schedl A, and differentiation. In: Veterinary Pathology, 6th ed. Williams & Behringer RR. Sox9 in testis determination. Ann NY Acad Wilkins Inc., Baltimore, MD. 1150–1153. 1997. Sci. 1061: 9–17. 2005. 2. Melniczek JR, Dambach D, Prociuk U, Jezyk PF, Henthorn 17. Meyers-Wallen VN. Sry and Sox9 expression during canine PS, Patterson DF, and Giger U. Sry-negative XX sex gonadal sex determination assayed by quantitative reverse reversal in a family of Norwegian Elkhounds. J Vet Intern transcription-polymerase chain reaction. Mol Reprod Dev. Med. 13: 564–569. 1999. 65: 373–381. 2003. 3. Meyers-Wallen VN and Patterson DF. XX sex reversal in 18. Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, the American cocker spaniel dog: phenotypic expression and Chaboissier MC, Poulat F, Behringer RR, Lovell-Badge R, inheritance. Hum Genet. 80: 23–30. 1988. and Capel B. Fgf9 and Wnt4 act as antagonistic signals to 4. Selden JR, Wachtel SS, Koo GC, Haskins ME, and Patterson regulate mammalian sex determination. PLoS Biol. 4: e187. DF. Genetic basis of XX male syndrome and XX true 2006. hermaphroditism: evidence in the dog. Science. 201: 644– 19. Jeays-Ward K, Dandonneau M, and Swain A. Wnt4 is 646. 1978. required for proper male as well as female sexual Kobayashi, Fujiwara, Adachi et al. 115

development. Dev Biol. 276: 431–440. 2004. treatment with letrozole after the thermosensitive period for 20. Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, sex determination. J Exp Zool. 290: 490–497. 2001. Swain A, and Capel B. Follistatin operates downstream of 25. Vaillant S, Dorizzi M, Pieau C, and Richard-Mercier N. Sex Wnt4 in mammalian ovary organogenesis. Dev Dyn. 230: reversal and aromatase in chicken. J Exp Zool. 290: 727– 210–215. 2004. 740. 2001. 21. de Rooster H, Vercauteren G, Gortz K, Saunders J, Polis I, 26. Kothapalli K, Kirkness E, Pujar S, Van Wormer R, and and Rijsselaere T. True hermaphroditism in six female Meyers-Wallen VN. Exclusion of candidate genes for canine littermates after administration of synthetic to a SRY-negative XX sex reversal. J Hered. 96: 759–763. 2005. pregnant bitch. Reprod Domest Anim. 41: 22–26. 2006. 27. Kothapalli KS, Kirkness EF, Pujar S, and Meyers-Wallen 22. Jordan BK, Shen JH, Olaso R, Ingraham HA, and Vilain E. VN. Exclusion of WT1 as a candidate gene for canine SRY- Wnt4 overexpression disrupts normal testicular vasculature negative XX sex reversal. Anim Genet. 35: 466–467. 2004. and inhibits testosterone synthesis by repressing 28. Kothapalli KS, Kirkness EF, Vanwormer R, and Meyers- steroidogenic factor 1/beta-catenin synergy. Proc Natl Acad Wallen VN. Exclusion of DMRT1 as a candidate gene for Sci USA. 100: 10866–10871. 2003. canine SRY-negative XX sex reversal. Vet J. 171: 559–561. 23. Kroon FJ, Munday PL, Westcott DA, Hobbs JP, and Liley 2006. NR. Aromatase pathway mediates sex change in each 29. Pujar S, Kothapalli KS, Kirkness E, Van Wormer RH, and direction. Proc Biol Sci. 272: 1399–1405. 2005. Meyers-Wallen VN. Exclusion of Lhx9 as a candidate gene 24. Belaid B, Richard-Mercier N, Pieau C, and Dorizzi M. Sex for Sry-negative XX sex reversal in the American cocker reversal and aromatase in the European pond turtle: spaniel model. J Hered. 96: 452–454. 2005.