Developmental Changes of Müllerian and Wolffian Ducts in Domestic Cat Fetuses

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1 Exp. Anim. 58(1), 41 45, 2009 Note Developmental Changes of Müllerian and Wolffian Ducts in Domestic Cat Fetuses Tomo INOMATA 1), Hiroyoshi NINOMIYA 1), Katsuyasu SAKITA 1), Naomi KASHIWAZAKI 2), Junya ITO 2), Makoto ARIGA 3), and Seiya INOUE 4) 1) Department of Laboratory Animal Science, School of Veterinary Medicine, Azabu University, Sagamihara , 2) Department of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara , 3) Ariga Animal Hospital, Kajigaya, Yokohama , and 4) ARK Resource Co., Ltd., Nakahara, Kumamoto , Japan Abstract: At 0.8 cm of crown-rump length (CRL) in sexually undifferentiated cat fetuses, the Wolffian duct is already observable on the ventro-lateral side of the mesonephros. At 1.2 cm CRL, the anlage of the Müllerian duct growing caudally in close parallel with the Wolffian duct was first observed. At 2.8 cm CRL in sexually differentiated fetuses, the Müllerian duct reached the urogenital sinus but remained indiscernible. Subsequently, at 3.2 cm CRL, regression of the upper part of the Müllerian duct was visible in males, while both ducts continued to grow in females. This suggests that the Müllerian inhibiting substance is produced before 3.2 cm CRL. At 7.0 cm CRL, male and female Wolffian ducts were reduced in diameter by about 50%, accompanied by involution of the mesonephros. Thereafter, the Wolffian duct was retained in the male; however, at 8.5 cm CRL in the female, the Wolffian duct was greatly reduced, by about 80% in diameter, then disappeared completely at 9.0 cm CRL. Key words: cat, Müllerian duct, Wolffian duct The mammalian fetal testis has a very important role in controlling the masculinization of the undifferentiated fetal urogenital tract. The fetal testis secretes testosterone that stimulates both the development of the Wolffian (mesonephric) duct and the masculinization of the external genitalia [5, 10]. The testis also secretes the Müllerian inhibiting substance which induces regression of the Müllerian (paramesonephric) duct [1, 4, 11]. On the other hand, the fetal ovary does not influence early development of the urogenital tracts [5, 10]. These morphological and physiological data have been accumulated for many species such as mice, rats, guinea pigs, rabbits, pigs, sheep, and cattle. However, data on the development of the feline urogenital tract is scarce. The objectives of the present study were to determine at what period the sex difference is observable in the development of Wolffian and Müllerian ducts in domestic cat fetuses. Various information was taken into account from the basic background of normal sexual differentiation in other investigations such as male and female pseudohermaphroditism [2, 6], persistent Müllerian duct syndrome [9], testicular feminization [7, 8], and dysgenesis of reproductive tracts [3] in felines. All procedures used in this study were in accordance with the guidelines approved by the Azabu University Animal Experiment Committee. (Received 12 May 2008 / Accepted 10 July 2008) Address corresponding: T. Inomata, Department of Laboratory Animal Sciences, School of Veterinary Medicine, Azabu University, Fuchinobe, Sagamihara, Kanagawa , Japan

2 42 T. Inomata, ET AL. Müllerian and Wolffian duct diameters (μm) Wolffian duct (sex indifferent) Female Wolffian duct Male Wolffian duct Female Müllerian duct Male Müllerian duct Crown-rump length (cm) Fig. 1. Changes in the diameter of Müllerian and Wolffian ducts in sex indifferent and male and female cat fetuses. A dotted line shows the disappearance of the male Müllerian and female Wolffian ducts. Thirty-two domestic cat fetuses were obtained from animal hospitals at a time when their mothers were subjected to surgical contraception as birth control. Fetuses ranging from 0.8 to 10.5 cm in crown-rump length (CRL), were used in this study. After measuring, the fetuses were sacrificed by decapitation under cold anesthesia with ice cubes. Fetuses less than 5 cm CRL were fixed in a 10% (v/v) formalin solution. Larger fetuses were treated as follows. The cranial half of the body was cut off at the diaphragm level and the liver and intestines were removed prior to fixation. Then, the formalin-fixed materials were transferred into Bouin s fixative. All materials were dehydrated in a graded series of ethanol and embedded in paraffin. Then, fetuses smaller than the 3.5 cm CRL were serially sectioned at 6 µm. Fetuses larger than 4.5 cm CRL were sectioned at 6 µm in every 60, 120, or 300 µm. The sections were stained with hematoxylin and eosin. The diameter of the Müllerian ducts was measured at the middle level of the gonads and the diameter of the Wolffian ducts was measured at the level where the mesonephroinguinal ligament is attached near the caudal end of the mesonephros. Diameters of the ducts on both sides were averaged for each fetus. The sex of the fetuses was distinguishable by histological inspection of their gonads. The main criterion for determination of the males was the formation of anlage, 10 which are seminiferous tubules observed in the gonads, in fetuses of 2.2 cm CRL, as described in the results. The sexual differentiation of the external genitalia was first observed in male fetuses of 3.2 cm CRL, when an anogenital raphe appeared and the caudal portion of the genital swellings moved and fused to each other as the scrotum in the region caudal to the genital tubercle. In females, however, these features were not observable. The development patterns of the Müllerian and Wolffian ducts are expressed by their diameters as shown in Fig. 1. Indifferent sexual stage, 0.8 cm CRL: The Wolffian duct situated in the ventro-lateral side of each Wolffian body (mesonephros) is visible (Fig. 2), and has already come into contact with the urogenital sinus. The ducts are lined with a layer of cuboidal epithelial cells. The cranial tips are situated immediately beneath the mesothelium at the cranial end of the mesonephros, near the site caudal to mesonephro diaphragmatic ligament attachment. Cells at this portion of the mesothelium are thick, compact, and densely packed. The Wolffian duct in both sexes increases in diameter from this stage until 3.6 cm CRL. The Müllerian duct is not visible at this stage. 1.2 cm CRL: On the craniolateral surface of each mesonephros, the anlage of the Müllerian duct, called the Müllerian groove, is evident (Fig. 3-A). Somewhat caudal to this groove the actual Müllerian duct is visible (Fig. 3-B). Caudally, a little closer to the apical portion of the Müllerian duct, a cord-like structure is visible. The cord-like aggregate of cells comes into close contact with the Wolffian duct just beneath the surface of the mesonephros (Fig. 3-C). The tip of the duct has reached the upper middle area caudal to the mesonephros. The Müllerian duct remains non-patent until 3.2 cm CRL. Differentiated stage of the gonads, 2.2 cm CRL: By this stage, anlages of seminiferous tubules are observed in the gonads (Fig. 4). The fetus can be identified as a male by these findings. In female fetuses, these findings are not observable (Fig. 5). The upper part of the Müllerian ducts provide a lumen lined with a single columnar epithelium (Fig. 6-A). The ducts extend caudally, beyond the posterior end of the mesonephros. The duct is devoid of lumen towards the caudal end that exhibits a cord-like structure as noted in the previous stage (Fig. 6-B). 2.8 cm CRL: The Müllerian duct in both sexes has

3 MÜLLERIAN AND WOLFFIAN DUCTS IN CAT FETUSES 43 a: aorta, i: intestine, k: kidney, m: Müllerian duct, md: mesonephrodiaphragmatic ligament, ml: mesonephroinguinal ligament, mg: Müllerian groove, o: ovary, s: seminiferous tubules, t: testis, w: Wolffian duct, wb: Wolffian body (mesonephros). Fig. 2. A fetus of 0.8 cm CRL (indifferent sexual stage): The Wolffian duct (w) situated in the ventro-lateral side of each mesonephros (Wolffian body; wb) is visible. The ducts are lined with a layer of cuboidal epithelial cells. The cranial tips are situated immediately beneath the mesothelium at the cranial end of the mesonephros, near the site caudal to mesonephro diaphragmatic ligament (md) attachment. Cells at this portion of the mesothelium are thick, compact, and densely packed. The Müllerian duct is not visible at this stage. Bar=125 µm. Fig. 3. A fetus of 1.2 cm CRL: On the ventro-lateral surface of each mesonephros (wb), the anlage of the Müllerian duct, called the Müllerian groove (mg) evident (A, Bar=250 µm). Somewhat caudal to this groove, the Müllerian duct (m) is visible (B, Bar=100 µm). A little closer, caudal to the apical portion of the Müllerian duct, a cord-like structure can be seen. The cord-like aggregate of cells nearly makes contact with the Wolffian duct (w) just beneath the surface of the mesonephros (C, Bar=100 µm). Fig. 4. A male fetus of 2.2 cm CRL (differentiated stage of the gonad): Anlages of seminiferous tubules (s) are observable in the gonads (testis; t). This indicates that this fetus is male. The kidney (k) is observable beside the mesonephros (wb). The Müllerian duct (m) is evident in the ventro-lateral side of the mesonephros. Bar=500 µm. Fig. 5. A female fetus of 2.2 cm CRL: Anlages of seminiferous tubules as seen in the male fetus are not observed in the gonads (ovary; o). This fetus is identified as a female. The kidney (k) is observable beside the mesonephros (wb). The Müllerian duct (m) is evident in the ventrolateral side of the mesonephros. Bar=500 µm.

4 44 T. INOMATA, ET AL. i: intestine, m: Müllerian duct, ml: mesonephroinguinal ligament, w: Wolffian duct, wb: Wolffian body (mesonephros). Fig. 6. A male fetus of 2.2 cm CRL: The upper part of the Müllerian duct (m) provides a lumen lined with a single columnar epithelium (A, Bar=200 µm). Caudally, both sides of the Müllerian and Wolffian ducts come close to each other in the midline (B, Bar=100 µm). The caudal end of the Müllerian duct (right duct) is devoid of the lumen. The duct exhibits cord-like structures (see arrow) which nearly come into contact with the Wolffian duct as noted in the previous stage. Fig. 7. A male fetus of 3.2 cm CRL (differentiated stage of the external genitalia): The Müllerian duct (m) shows signs of degeneration at the mesonephroinguinal ligament (ml) attachment. The cells of the duct contain small basophilic granules, and there are several sites where segments of the ducts have disappeared, which are most evident cranially. Bar=100 µm. Fig. 8. A female fetus of 3.0 cm CRL: the Müllerian (m) and the Wolffian (w) ducts at the mesonephroinguinal ligament attachment (ml) are well developed. Bar=100 µm. Fig. 9. A male fetus of 7.0 cm CRL: The Wolffian duct (w) has diminished in diameter as a result of the degeneration of the mesonephros. The Müllerian duct has completely disappeared. Bar=200 µm. Fig. 10. A female fetus of 7.0 cm CRL: The Müllerian duct (m) grows slightly. The Wolffian duct (w) has diminished in diameter as is described at the same stage of the male fetus. Bar=200 µm. Fig. 11. A male fetus of 8.5 cm CRL: The male Wolffian duct continues to maintain its size. The duct becomes an epididymal duct. Bar= 200 µm. Fig. 12. A female fetus of 8.5 cm CRL: The Müllerian duct (m) is well developed. The Wolffian duct (w) has radically shrunk in diameter. Bar= 200 µm.

5 MÜLLERIAN AND WOLFFIAN DUCTS IN CAT FETUSES 45 reached the urogenital sinus but the ducts remain indiscernible. The diameter of the Müllerian duct is identical in both sexes until this stage. Differentiated stage of the external genitalia, 3.2 cm CRL: Müllerian ducts are fully patent in both sexes. The diameter of the Müllerian ducts is smaller in the male (Fig. 7) than in the female (Fig. 8). Male Müllerian ducts show signs of degeneration. The cells of the duct contain small basophilic granules, and there are several sites where segments of the ducts have disappeared, most evident cranially. These events occur from the cranial to caudal regions. Finally, in fetuses of 5.0 cm CRL only the cranial tip (Müllerian groove) and caudal end (prostatic utriculus) remain. In the female the ducts grow slightly until 7.0 cm CRL. Then, the duct drastically grows. 4.5 cm CRL: The mesonephros has started to degenerate by this stage. As a result of this degeneration the Wolffian ducts diminish in diameter and this reduction continues to occur in fetuses of 7.0 cm CRL in both males (Fig. 9) and females (Fig. 10). By this stage, the prostatic buds are visible near the urogenital sinus in males. 8.5 cm CRL: The male Wolffian duct continues to maintain its size until the end of gestation (Fig. 11). At this stage, all the mesonephros has degenerated except in the male Wolffian tubules which are forming epididymal ducts. The Wolffian ducts have shrunken extremely in diameter by this stage in the female (Fig. 12). Then, the female Wolffian duct all but disappears except for its cranial tip (epoophoron). The Müllerian ducts are first evident in feline fetuses of 1.2 cm CRL of both males and females. A lumen has formed everywhere except in the caudal solid-end of the ducts in fetuses of 1.2 to 2.8 cm CRL. At 3.2 cm CRL, the ducts in both sexes are well developed and have reached the urogenital sinus. However, at the same stage in the male, the upper part of the duct has started to degenerate. It is well established that Müllerian ducts are degenerated by a testicular factor which is different from testosterone [4, 5]. This factor is called the Müllerian inhibiting substance and is synthesized by fetal sustentacular (Sertoli) cells [1, 4, 11]. In the present study, the diameters of Müllerian ducts were not different between males and females until fetal lengths of 2.8 cm CRL. At this stage the ducts had reached the urogenital sinus but the ducts remained non-patent. At 3.2 cm CRL, the Müllerian ducts of all fetuses are fully patent, but the male ducts are degenerating. Based on these signs, it can be concluded that, in felines, the Müllerian duct develops a sensitivity to the Müllerian-inhibiting substance before a fetal length of 3.2 cm CRL. In other parts of the urogenital tracts such as the external genitalia, the masculinization of sexually undifferentiated fetuses is attributable to androgen secretion from the Leydig cells [5, 10, 12]. Regarding the maintenance and development of the Wolffian duct, in both sexes it begins to decrease in diameter after fetal length has attained 3.6 cm CRL. This phenomenon is accompanied by the involution of the mesonephros which starts at about the same time. At 7.0 cm CRL, the male and female Wolffian ducts have shrunk in diameter even more and the mesonephros has largely degenerated. However, male Wolffian ducts begin to develop into the epididymal duct at about this stage. At 9.0 cm CRL, the female Wolffian duct has diminished greatly. Thereafter, the female duct completely disappears. The male Wolffian duct continues to remain the same size. These findings suggest that the male Wolffian duct begins to require androgen in order to maintain and develop itself at a fetal lenght of 7.0 cm CRL. References 1. Blanchard, M.G. and Josso, N Pediatr. Res. 8: Bredal, W.P., Thoresen, S.I., Kvellestad, A., and Lindblad, K J. Small Anim. Pract. 38: Hakala, J.E Mod. Vet. Pract. 65: Josso, N J. Clin. Endocrinol. Metab. 32: Jost, A Recent Prog. Horm. Res. 8: Knighton, E.L J. Am. Vet. Med. Assoc. 225: Lawhorn, B J. Am. Vet. Med. Assoc. 195: Meyers-Wallen, V.N., Wilson, J.D., Griffin, J.E., Fisher, S., Moorhead, P.H., Goldschmidt, M.H., and Haskins, M.E J. Am. Vet. Med. Assoc. 195: Meyers-Wallen, V.N J. Reprod. Fertil. Suppl. 47: Price, D. and Ortiz, E pp In: Organogenesis, 1st ed. (DeHaan, R.L. ed.), Holt Rinehart and Winston, New York. 11. Tran, D. and Josso, N Endocrinology 111: Weisz, J. and Ward, I Endocrinology 106: