THE JOURNAL OF EXPERIMENTAL ZOOLOGY 262:454-457 (1992)
RAPID COMMUNICATION
Steroid Hormone-Induced Male Sex Determination in an Amniotic Vertebrate THANE WIBBELS, JAMES J. BULL, AND DAVID CREWS Department of Zoology, Institute of Reproductive Biology, University of Texas at Austin, Austin, Texas 78712
ABSTRACT In many reptiles, sex is determined by the temperature a t which the eggs are incubated (i.e., temperature-dependent sex determination, or TSD). Past studies have shown that exogenous steroid hormones can override the effects of temperature and induce female sex determination. However, past attempts to induce male sex determination have consistently failed. In the present study, sex determination was studied in a turtle with TSD. By utilizing a n incubation temperature regimen that resulted in approximately a 1:lsex ratio in the control group, sex determination was shown to be sensitive to both exogenous androgen and estrogen treatments: androgen induced the production of male hatchlings, whereas estrogen induced the production of female hatchlings. This is the first report of an amniotic vertebrate in which a n exogenous steroid hormone induces male sex determination. o 1992 Wiley-Liss, Inc.
The mode of sex determination varies among reptiles and includes species with genotypic sex determination and others with temperature-dependent sex determination (TSD)(Bull, '80). Heteromorphic chromosomes have not been detected in reptiles with TSD and it is unknown if these species possess a genotypic sex which is obscured by the effects of incubation temperature (Bull, '83). Past studies of reptiles with TSD have shown that administration of exogenous estrogens, and to a lesser extent testosterone, can induce embryos to develop as females even if eggs are incubated at male-producing temperatures (reviewed by Raynaud and Pieau, 1985; also see Gutzke and Bull, '86; Bull et al., '88; Crews et al., '89, '91; Wibbels and Crews, '92). It has been suggested that the feminizing action of steroid hormones is estrogen-specific and that the effects of testosterone may be mediated via aromatization (Crews et al., '89). In these previous studies, however, steroid hormone treatments failed to induce the production of males from eggs incubated at female-producing temperatures. This suggests that steroid hormones are capable of driving sex determination only in the female direction. To address this hypothesis, we have incubated eggs of the red-eared slider turtle, k c h e m y s scriptu, in a manner that produced a relatively equal proportion of male and female hatchlings in the control group. Other groups of eggs received either androgen or estrogen treatments.
MATERIALS AND METHODS Freshly laid eggs from red-eared slider turtles were obtained commercially (Robert Kliebert, Ham0 1992 WILEY-LISS, INC.
mond, LA). After transport to our laboratory, eggs were placed in containers with moistened vermiculite (vermiculite:water, 1:2). Embryonic development was monitored by candling and by dissecting 2-4 eggs approximately twice a week t o verify specific developmental stages (Yntema, '68). Previous studies with this species indicate that a continuous incubation temperature of 26°C produces all male hatchlings whereas an incubation temperature of 32°C produces all female hatchlings (Bull et al., '82; Crews et al., '91). A recent study indicates that sex determination in this turtle is sensitive t o temperature from approximately stage 15 through stages 19-21, depending upon the specific incubation temperature (Wibbels et al., '91b). Furthermore, the sexual fate of all embryos incubated at a male-producing temperature (26°C) becomes irreversibly male by approximately stage 21, whereas the sexual fate of all embryos incubated at a female-producingtemperature (31°C)becomes irreversibly female by approximately stages 19-20 (Wibbels et al., '91b). The experimental design adopted for the current study was based, in part, on a preliminary study in our laboratory. In that study, eggs were incubated at 26°C until approximately stage 20. They then received a hormone treatment (100 pg of DHT in 5 pl of ethanol) or a control treatment (5 pl of ethanol) and were shifted to 31°C for the remainder of their incubation. Upon hatching, Fkceived March 5,1991; revision accepted May 31,1991.
STEROID-INDUCED MALENES!3 IN AMNIOTIC VERTEBRATE
68.4% of the control group were males (6 females:13 males), whereas 93.8% of the DHT group were males (1female:15 males). In the current study, approximately 100 eggs, which had all been laid during the same 48-hr period, were incubated at 26°C until approximately stage 19.5 (a point late in the temperature-sensitive period of this turtle) (Wibbelset al., '91b). Eggs were then randomized into four groups (25 eggs/group) and each egg received either a hormone or control treatment. All four groups of eggs received either hormone or control treatments during the same 1-hr period. Hormone treatments consisted of either 200 pg 17~-hydroxy-5~-androstan-3-one (DHT),100 pg DHT, or 10 pg 17P-estradiol (E2),suspended in 5 pl of 95% ethanol. The dosages chosen for each steroid were based on previous studies with turtles (Crewset al., '89, '91). Control treatment consisted of 5 pl of 95% ethanol. All treatments were applied topically to the vascularized portion of the upper shell (Crews et al., '91). Eggs were then placed in a 32°C incubator until hatch. Consistent with past results (Crews et al., ,911, mortality during the remainder of the incubation period was limited to only a small number of developingembryos (1-3 per treatment group). Hatchlings were euthanized and their gonadal sex and developmental status of the oviducts assessed by examination of the reproductive tracts under a dissection microscope. Additionally, a single gonad from each hatchling was histologically processed (Humason, '72). Histological data verified the initial visual assessment in all cases.
loo 90
455
i
-
80 -
0
Control
DHT 100 /Jug
DHT 200 p,g
E2
10 I*g
Fig. 1. Percentage of male hatchlings produced in response to DHT and estradiol-17P (Ez)treatments.
oviducts whereas the majority of the males (77%) either lacked oviducts (n = 9) or had regressed oviducts (n = 8). Of the remaining males, three had normal appearing right oviducts but regressed left oviducts and two had normal oviducts bilaterally. In the group that received 100 pg DHT, the two females had normal oviducts, whereas again the majority of males (80.5%)either lacked oviducts (n = 15) or had regressed oviducts (n = 2). Of the remaining males, two had regressed right oviducts and lacked left oviducts and two had normal oviRESULTS ducts bilaterally. All individuals receiving 10 pg The percentage of male hatchlings in each group E2 had normal oviducts (n = 22). The presence of is shown in Figure 1. The sex ratios of the groups normal oviducts in four males that received DHT exhibited significant variation (chi-square, P < treatments appears to contradict the standard par0.001) with androgen treatments resulting in sigadigm of sex determination and sexual differentinificantly more males than the control group (Fish'70). However, because the hormone ation (Jost, er's exact test, P < 0.001); estrogen treatment treatments were given late in the temperatureresulted in significantly more females than the consensitive period, oviductal regression may simply trol group (Fisher's exact test, P < 0.001). There be delayed in some individuals that received DHT. were no histological differences detected in the tesAlternatively, DHT may have altered the producticular or ovarian nature of gonads from hormoneor activity of Mullerian inhibiting substance. tion treated hatchlings versus male and female control hatchlings (Fig. 2). Hermaphroditic gonads were DISCUSSION not observed. The presence or absence of the oviducts, an accesThe results indicate that sex determination in sory sex structure, was consistent with gonad type. these embryos was bipotentially sensitive to sex steOf the control individuals, all females had normal roids. The reason why steroid hormones have failed oviducts and of the 12 males, ten lacked oviducts to induce male sex determination in past studies and two had regressed oviducts. In the group that is unknown, but it could relate to the effects of conreceived 200 pg DHT, the two females had normal tinuous incubation at female-producing tempera-
456
T. WIBBELS ET AL.
Fig. 2. Gonads from hatchlings which received either a control treatment (5 p1 of ethanol) or a steroid hormone treatment (in 5 p1 of ethanol) during the middle third of incubation. a: Control male; b: individual that received 200 pg DHT; c: control female; d: individual that received 10 pg estradiol-17P.
tures. A recent study indicates that incubation temperature can significantly affect the feminizing potency of estrogen (Wibbels et al., '91a). The protocol used in the present study, in which eggs were incubated at a male-producing temperature until stage 19.5 and then shifted to a female-temperature, may have initiated physiological events in the male sex determination cascade which rendered the sex determination system sensitive to DHT stimulation. However, if such events occurred, the results clearly show that it did not irreversibly affect the sexual fate of embryos since estrogen treatments resulted in the production of all females. Alternatively, it may be that incubation at a male-producing temperature maintains the embryo in a sexually labile state until late in the temperature-sensitive period, whereas continuous incubation at a female-producing temperature may initiate early events in the
female sex determination cascade which prevent or mask the effects of DHT. Of particular interest, recent data from our laboratory suggests that incubation at an intermediate temperature, producing a 1:l sex ratio in control groups, results in embryos in which sex determination is bipotentially sensitive to steroid hormones (Wibbels and Crews, unpublished data. The specific mechanism by which sex steroids affect sex determination is unknown. However, the results are consistent with the hypothesis that the masculinizing or feminizing effects are mediated through steroid-specific receptors. This is the first report suggesting that estrogen and androgen receptors which mediate opposite effects on sex determination may be present. Alternatively, the high dosages of DHT could have antagonized the effects of endogenous estrogen by competing for estrogen receptor. It has been hypothesized that endogenous estrogen could be an important factor naturally involved in female sex determination of reptiles with TSD (Crews et al., '89). Regardless, the ability of DHT to induce male sex determination provides an additional avenue by which this system can be manipulated in future studies investigating the molecular basis of TSD. The findings of previous studies that sex steroids induce only female sex determination in reptiles with TSD were consistent with the concept of a "default sex" model for sex determination (Jost, ,701, with exogenous estrogen either blocking or stimulating the physiological cascade that would lead to the induced sex. The present results, however, suggest the possibility of active physiological cascades leading t o each sex. It will be of interest to determine if the male and female components of sex determination can be simultaneously stimulated by a combination of estrogen and androgen treatments to produce hermaphroditic gonads in this TSD system. It has long been known that steroid hormones affect sex determination in many fishes and amphibians (Reinboth,'75; Witschi, '39;Witschi and Chang, '50; Witschi and Dale, '62; Yamamoto, '621, with the production of males or females dependent upon the specific steroid treatment. In birds and marsupial mammals, steroid hormones can exert partial masculinizing or feminizing effects on the gonads, but full sex reversals have not been achieved (Benoit, '23;Benoit, '50; Burns, '55, '56; Domm, '24, Domm, '39; Fadem and Tesoriero, '86; Maraud et al., '87; Shaw et al., '88; VanTienhoven, '57). (See note added in proof.) In eutherian mammals, steroid hormones do not appear to affect gonadal differentiation (Jost, '70). The results of the present study suggest that at
STEROID-INDUCED MALENESS IN AMNIOTIC VERTEBRATE
457
least some reptiles may represent a primitive or Domm, L.V. (1924) Sex reversal following ovariectomy in the fowl. Proc. SOC. Exp. Biol. Med., 22:28-35. transitional model for amniotic sex determination, Domm, L.V. (1939) Modifications of sex and secondary sexual with sex determination being bipotentially sensicharacters in birds. In: Sex and Internal Secretions. E. Allen, tive to steroid hormones. ed. Williams & Wilkins, Baltimore, pp. 227-327. Fadem, B.H., and J.V. Tesoriero (1986) Inhibition of testicular ACKNOWLEDGMENTS development and feminization of the male genitalia by neonatal estrogen treatment in a marsupial. Biol. Reprod., We would like to acknowledgethe technical assis34:771-776. tance of Anthony Alexander. This research was sup- Gutzke, W.H.N., and J.J. Bull (1986) Steroid hormones reverse ported by NIH grant HD-24976 and NIMH Research sex in turtles. Gen. Comp. Endocrinol. 64:368-372. Scientist Award 00135 to D.C.,by NIH NRSA train- Humason, G.L. (1972) Animal Tissue Techniques. W.H. Freeman and Company, San Francisco. ing grant HD-07264 to the Institute of Reproductive Biology, and by NIH NRSA HD-07319-01A1 Jost, A. (1970) Hormonal factors in sex differentiation of the mammalian foetus.Philos. Trans.R. Land. [Bi01.1,259:119-130. to T.W. Maraud, R., 0. Vergnaud, and M. Rashedi (1987) Structure of
NOTE ADDED IN PROOF Since acceptance of this paper, Elbrecht and Smith (Science 255:467-470) report that administration of a specific aromatase inhibitor induces testis formation in genotypic female chickens. LITERATURE CITED Benoit, J. (1923) Transformation experimentale du sexe par ovari‘ectomie precoce chez la Poule domestique. C. R. Acad. Sci., 177:1074-1077. Benoit, J. (1950) Diffbrenciation sexuelle chez les 0 seaux au cours du dheloppement normal et de l’intersexualitk experimentale par ovariectomie.Arch. Anat. Microsc. Morphol. Exp., 39:395-410. Bull, J.J. (1980) Sex determination in reptiles. Q. Rev. Biol., 55:3-21. Bull, J.J. (1983) Evolution of Sex Determining Mechanisms. BenjamidCummings Publishing Company, London. Bull, J.J.,R.C. Vogt, and C.J. McCoy (1982) Sex determining temperatures in turtles: A geographic comparison. Evolution, 36:326-332. Bull, J.J., W.H.N. Gutzke, and D. Crews (1988) Sex reversal by estradiol in three reptilian orders. Gen. Comp. Endocrinol., 70:425-428. Burns, R.K. (1955) Experimental reversal of sex in the gonads of the opossum Didelphis uirginiana. Proc. Natl. Acad. Sci. U.S.A., 41 :669-676. Burns, R.K. (1956) Hormones vs. constitutional factors in the growth of embryonic sex primordia in the sex primordia in the oppossum. Am. J. Anat., 98:35-68. Crews, D., T.Wibbels, and W.H.N. Gutzke (1989) Action of sex steroid hormones on temperature-induced sex determination in the snapping turtle (Chelydra serpentinu). Gen. Comp. Endocrinol., 76: 159- 166. Crews, D., J.J. Bull, and T. Wibbels (1991) Estrogen and sex reversal in turtles: A dose-dependentphenomenon. Gen. Comp. Endocrinol., 81:357-364.
the right testis in sexually mature genetically female fowl experimentally masculinized during embryonic life and submitted to a posthatching left castration. Gen. Comp. Endocrinol., 68:208-215. Raynaud, A., and C. Pieau (1985) Embryonic development of the genital system. In: Biology ofthe Reptilia, Vol. 15, Development B. C. Gans and F. Billett, eds. John Wiley & Sons, New York, pp. 149-300. Reinboth, R. (1975) Spontaneous and hormone induced sexinversion in wrasse. Pubbl. Stn. Zool. Napoli (II),39[suppl]: 550-573. Shaw, G., M.B. Renfree, R.V. Short, and W.-S. 0 (1988) Experimental manipulation of sexual differentiation in Wallaby pouch young treated with exogenous steroids. Development, 104:689-701. VanTienhoven, A. (1957) A method of “controlling sex” by dipping of eggs in hormone solution. Poult. Sci., 36:628-632. Wibbels, T., J.J.Bull, and D. Crews (1991a) Synergism between temperature and estradiol: A common pathway in sex determination? J . Exp. Zool. 260:130-134. Wibbels, T,, J.J. Bull, and D. Crews (1991b) Chronology and morphology of temperature-dependent sex determination. J . EXP. ZOO^. 260:371-381. Wibbels, T. and D. Crews (1992) Specificity of steroid hormoneinduced sex determination in a turtle. J. Endocrinol. 133: 121-129. Witschi, E. (1939) Modification of the development of sex in lower vertebrates. In: Sex and Internal Secretions. C.H. Danforth and E.A. Doisy, eds. Williams and Wilkins Co., Baltimore, pp. 145-226. Witschi, E., and C.Y. Chang (1950) Cortisone induced transformation of ovaries into testes in larval frogs. SOC. Exp. Biol. Med., 75:715-718. Witschi, E., and E. Dale (1962) Steroid hormones at early developmental stages of vertebrates. Gen. Comp. Endocrinol. 1 (suppl):356-361. Yamamoto, T. (1962) Hormonic factors affecting sex differentiation in fish. Gen. Comp. Endocrinol, 1 (suppl):341-345. Yntema, C.L. (1968) A series of stages in the embryonic development of Chelydra serpentina. J. Morphol., 125:219-252.
RAPID COMMUNICATION
Steroid Hormone-Induced Male Sex Determination in an Amniotic Vertebrate THANE WIBBELS, JAMES J. BULL, AND DAVID CREWS Department of Zoology, Institute of Reproductive Biology, University of Texas at Austin, Austin, Texas 78712
ABSTRACT In many reptiles, sex is determined by the temperature a t which the eggs are incubated (i.e., temperature-dependent sex determination, or TSD). Past studies have shown that exogenous steroid hormones can override the effects of temperature and induce female sex determination. However, past attempts to induce male sex determination have consistently failed. In the present study, sex determination was studied in a turtle with TSD. By utilizing a n incubation temperature regimen that resulted in approximately a 1:lsex ratio in the control group, sex determination was shown to be sensitive to both exogenous androgen and estrogen treatments: androgen induced the production of male hatchlings, whereas estrogen induced the production of female hatchlings. This is the first report of an amniotic vertebrate in which a n exogenous steroid hormone induces male sex determination. o 1992 Wiley-Liss, Inc.
The mode of sex determination varies among reptiles and includes species with genotypic sex determination and others with temperature-dependent sex determination (TSD)(Bull, '80). Heteromorphic chromosomes have not been detected in reptiles with TSD and it is unknown if these species possess a genotypic sex which is obscured by the effects of incubation temperature (Bull, '83). Past studies of reptiles with TSD have shown that administration of exogenous estrogens, and to a lesser extent testosterone, can induce embryos to develop as females even if eggs are incubated at male-producing temperatures (reviewed by Raynaud and Pieau, 1985; also see Gutzke and Bull, '86; Bull et al., '88; Crews et al., '89, '91; Wibbels and Crews, '92). It has been suggested that the feminizing action of steroid hormones is estrogen-specific and that the effects of testosterone may be mediated via aromatization (Crews et al., '89). In these previous studies, however, steroid hormone treatments failed to induce the production of males from eggs incubated at female-producing temperatures. This suggests that steroid hormones are capable of driving sex determination only in the female direction. To address this hypothesis, we have incubated eggs of the red-eared slider turtle, k c h e m y s scriptu, in a manner that produced a relatively equal proportion of male and female hatchlings in the control group. Other groups of eggs received either androgen or estrogen treatments.
MATERIALS AND METHODS Freshly laid eggs from red-eared slider turtles were obtained commercially (Robert Kliebert, Ham0 1992 WILEY-LISS, INC.
mond, LA). After transport to our laboratory, eggs were placed in containers with moistened vermiculite (vermiculite:water, 1:2). Embryonic development was monitored by candling and by dissecting 2-4 eggs approximately twice a week t o verify specific developmental stages (Yntema, '68). Previous studies with this species indicate that a continuous incubation temperature of 26°C produces all male hatchlings whereas an incubation temperature of 32°C produces all female hatchlings (Bull et al., '82; Crews et al., '91). A recent study indicates that sex determination in this turtle is sensitive t o temperature from approximately stage 15 through stages 19-21, depending upon the specific incubation temperature (Wibbels et al., '91b). Furthermore, the sexual fate of all embryos incubated at a male-producing temperature (26°C) becomes irreversibly male by approximately stage 21, whereas the sexual fate of all embryos incubated at a female-producingtemperature (31°C)becomes irreversibly female by approximately stages 19-20 (Wibbels et al., '91b). The experimental design adopted for the current study was based, in part, on a preliminary study in our laboratory. In that study, eggs were incubated at 26°C until approximately stage 20. They then received a hormone treatment (100 pg of DHT in 5 pl of ethanol) or a control treatment (5 pl of ethanol) and were shifted to 31°C for the remainder of their incubation. Upon hatching, Fkceived March 5,1991; revision accepted May 31,1991.
STEROID-INDUCED MALENES!3 IN AMNIOTIC VERTEBRATE
68.4% of the control group were males (6 females:13 males), whereas 93.8% of the DHT group were males (1female:15 males). In the current study, approximately 100 eggs, which had all been laid during the same 48-hr period, were incubated at 26°C until approximately stage 19.5 (a point late in the temperature-sensitive period of this turtle) (Wibbelset al., '91b). Eggs were then randomized into four groups (25 eggs/group) and each egg received either a hormone or control treatment. All four groups of eggs received either hormone or control treatments during the same 1-hr period. Hormone treatments consisted of either 200 pg 17~-hydroxy-5~-androstan-3-one (DHT),100 pg DHT, or 10 pg 17P-estradiol (E2),suspended in 5 pl of 95% ethanol. The dosages chosen for each steroid were based on previous studies with turtles (Crewset al., '89, '91). Control treatment consisted of 5 pl of 95% ethanol. All treatments were applied topically to the vascularized portion of the upper shell (Crews et al., '91). Eggs were then placed in a 32°C incubator until hatch. Consistent with past results (Crews et al., ,911, mortality during the remainder of the incubation period was limited to only a small number of developingembryos (1-3 per treatment group). Hatchlings were euthanized and their gonadal sex and developmental status of the oviducts assessed by examination of the reproductive tracts under a dissection microscope. Additionally, a single gonad from each hatchling was histologically processed (Humason, '72). Histological data verified the initial visual assessment in all cases.
loo 90
455
i
-
80 -
0
Control
DHT 100 /Jug
DHT 200 p,g
E2
10 I*g
Fig. 1. Percentage of male hatchlings produced in response to DHT and estradiol-17P (Ez)treatments.
oviducts whereas the majority of the males (77%) either lacked oviducts (n = 9) or had regressed oviducts (n = 8). Of the remaining males, three had normal appearing right oviducts but regressed left oviducts and two had normal oviducts bilaterally. In the group that received 100 pg DHT, the two females had normal oviducts, whereas again the majority of males (80.5%)either lacked oviducts (n = 15) or had regressed oviducts (n = 2). Of the remaining males, two had regressed right oviducts and lacked left oviducts and two had normal oviRESULTS ducts bilaterally. All individuals receiving 10 pg The percentage of male hatchlings in each group E2 had normal oviducts (n = 22). The presence of is shown in Figure 1. The sex ratios of the groups normal oviducts in four males that received DHT exhibited significant variation (chi-square, P < treatments appears to contradict the standard par0.001) with androgen treatments resulting in sigadigm of sex determination and sexual differentinificantly more males than the control group (Fish'70). However, because the hormone ation (Jost, er's exact test, P < 0.001); estrogen treatment treatments were given late in the temperatureresulted in significantly more females than the consensitive period, oviductal regression may simply trol group (Fisher's exact test, P < 0.001). There be delayed in some individuals that received DHT. were no histological differences detected in the tesAlternatively, DHT may have altered the producticular or ovarian nature of gonads from hormoneor activity of Mullerian inhibiting substance. tion treated hatchlings versus male and female control hatchlings (Fig. 2). Hermaphroditic gonads were DISCUSSION not observed. The presence or absence of the oviducts, an accesThe results indicate that sex determination in sory sex structure, was consistent with gonad type. these embryos was bipotentially sensitive to sex steOf the control individuals, all females had normal roids. The reason why steroid hormones have failed oviducts and of the 12 males, ten lacked oviducts to induce male sex determination in past studies and two had regressed oviducts. In the group that is unknown, but it could relate to the effects of conreceived 200 pg DHT, the two females had normal tinuous incubation at female-producing tempera-
456
T. WIBBELS ET AL.
Fig. 2. Gonads from hatchlings which received either a control treatment (5 p1 of ethanol) or a steroid hormone treatment (in 5 p1 of ethanol) during the middle third of incubation. a: Control male; b: individual that received 200 pg DHT; c: control female; d: individual that received 10 pg estradiol-17P.
tures. A recent study indicates that incubation temperature can significantly affect the feminizing potency of estrogen (Wibbels et al., '91a). The protocol used in the present study, in which eggs were incubated at a male-producing temperature until stage 19.5 and then shifted to a female-temperature, may have initiated physiological events in the male sex determination cascade which rendered the sex determination system sensitive to DHT stimulation. However, if such events occurred, the results clearly show that it did not irreversibly affect the sexual fate of embryos since estrogen treatments resulted in the production of all females. Alternatively, it may be that incubation at a male-producing temperature maintains the embryo in a sexually labile state until late in the temperature-sensitive period, whereas continuous incubation at a female-producing temperature may initiate early events in the
female sex determination cascade which prevent or mask the effects of DHT. Of particular interest, recent data from our laboratory suggests that incubation at an intermediate temperature, producing a 1:l sex ratio in control groups, results in embryos in which sex determination is bipotentially sensitive to steroid hormones (Wibbels and Crews, unpublished data. The specific mechanism by which sex steroids affect sex determination is unknown. However, the results are consistent with the hypothesis that the masculinizing or feminizing effects are mediated through steroid-specific receptors. This is the first report suggesting that estrogen and androgen receptors which mediate opposite effects on sex determination may be present. Alternatively, the high dosages of DHT could have antagonized the effects of endogenous estrogen by competing for estrogen receptor. It has been hypothesized that endogenous estrogen could be an important factor naturally involved in female sex determination of reptiles with TSD (Crews et al., '89). Regardless, the ability of DHT to induce male sex determination provides an additional avenue by which this system can be manipulated in future studies investigating the molecular basis of TSD. The findings of previous studies that sex steroids induce only female sex determination in reptiles with TSD were consistent with the concept of a "default sex" model for sex determination (Jost, ,701, with exogenous estrogen either blocking or stimulating the physiological cascade that would lead to the induced sex. The present results, however, suggest the possibility of active physiological cascades leading t o each sex. It will be of interest to determine if the male and female components of sex determination can be simultaneously stimulated by a combination of estrogen and androgen treatments to produce hermaphroditic gonads in this TSD system. It has long been known that steroid hormones affect sex determination in many fishes and amphibians (Reinboth,'75; Witschi, '39;Witschi and Chang, '50; Witschi and Dale, '62; Yamamoto, '621, with the production of males or females dependent upon the specific steroid treatment. In birds and marsupial mammals, steroid hormones can exert partial masculinizing or feminizing effects on the gonads, but full sex reversals have not been achieved (Benoit, '23;Benoit, '50; Burns, '55, '56; Domm, '24, Domm, '39; Fadem and Tesoriero, '86; Maraud et al., '87; Shaw et al., '88; VanTienhoven, '57). (See note added in proof.) In eutherian mammals, steroid hormones do not appear to affect gonadal differentiation (Jost, '70). The results of the present study suggest that at
STEROID-INDUCED MALENESS IN AMNIOTIC VERTEBRATE
457
least some reptiles may represent a primitive or Domm, L.V. (1924) Sex reversal following ovariectomy in the fowl. Proc. SOC. Exp. Biol. Med., 22:28-35. transitional model for amniotic sex determination, Domm, L.V. (1939) Modifications of sex and secondary sexual with sex determination being bipotentially sensicharacters in birds. In: Sex and Internal Secretions. E. Allen, tive to steroid hormones. ed. Williams & Wilkins, Baltimore, pp. 227-327. Fadem, B.H., and J.V. Tesoriero (1986) Inhibition of testicular ACKNOWLEDGMENTS development and feminization of the male genitalia by neonatal estrogen treatment in a marsupial. Biol. Reprod., We would like to acknowledgethe technical assis34:771-776. tance of Anthony Alexander. This research was sup- Gutzke, W.H.N., and J.J. Bull (1986) Steroid hormones reverse ported by NIH grant HD-24976 and NIMH Research sex in turtles. Gen. Comp. Endocrinol. 64:368-372. Scientist Award 00135 to D.C.,by NIH NRSA train- Humason, G.L. (1972) Animal Tissue Techniques. W.H. Freeman and Company, San Francisco. ing grant HD-07264 to the Institute of Reproductive Biology, and by NIH NRSA HD-07319-01A1 Jost, A. (1970) Hormonal factors in sex differentiation of the mammalian foetus.Philos. Trans.R. Land. [Bi01.1,259:119-130. to T.W. Maraud, R., 0. Vergnaud, and M. Rashedi (1987) Structure of
NOTE ADDED IN PROOF Since acceptance of this paper, Elbrecht and Smith (Science 255:467-470) report that administration of a specific aromatase inhibitor induces testis formation in genotypic female chickens. LITERATURE CITED Benoit, J. (1923) Transformation experimentale du sexe par ovari‘ectomie precoce chez la Poule domestique. C. R. Acad. Sci., 177:1074-1077. Benoit, J. (1950) Diffbrenciation sexuelle chez les 0 seaux au cours du dheloppement normal et de l’intersexualitk experimentale par ovariectomie.Arch. Anat. Microsc. Morphol. Exp., 39:395-410. Bull, J.J. (1980) Sex determination in reptiles. Q. Rev. Biol., 55:3-21. Bull, J.J. (1983) Evolution of Sex Determining Mechanisms. BenjamidCummings Publishing Company, London. Bull, J.J.,R.C. Vogt, and C.J. McCoy (1982) Sex determining temperatures in turtles: A geographic comparison. Evolution, 36:326-332. Bull, J.J., W.H.N. Gutzke, and D. Crews (1988) Sex reversal by estradiol in three reptilian orders. Gen. Comp. Endocrinol., 70:425-428. Burns, R.K. (1955) Experimental reversal of sex in the gonads of the opossum Didelphis uirginiana. Proc. Natl. Acad. Sci. U.S.A., 41 :669-676. Burns, R.K. (1956) Hormones vs. constitutional factors in the growth of embryonic sex primordia in the sex primordia in the oppossum. Am. J. Anat., 98:35-68. Crews, D., T.Wibbels, and W.H.N. Gutzke (1989) Action of sex steroid hormones on temperature-induced sex determination in the snapping turtle (Chelydra serpentinu). Gen. Comp. Endocrinol., 76: 159- 166. Crews, D., J.J. Bull, and T. Wibbels (1991) Estrogen and sex reversal in turtles: A dose-dependentphenomenon. Gen. Comp. Endocrinol., 81:357-364.
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