Repeated efforts to manipulate the primary sex ratio from unity traditionally have been unsuccessful (FIG. 1A). This includes not only breeding for biased sex ratios (e.g., Beamer and Whitten, 1991), but also by hormone manipulation of the embryo (van Tienhoven, 1957; Pincus and Hopkins, 1958). Unless there is differential survivorship, the one-to-one sex ratio will persist throughout life. There is an alternative sex determining mechanism in the higher or amniote (the reptiles, birds and mammals) vertebrates, one that is sensitive to the physical and physiological environment (FIG. 1B). In environmental sex determination, variation in the environment influences profoundly the primary sex ratio.
The fundamental difference between the mechanisms underlying genotypic sex determination (GSD) and environmental sex determination lies in the trigger that initiates sex determination; in the former, gonadal sex is determined at fertilization by the pairing of sex chromosomes, whereas in the latter, gonadal sex is determined much later in embryogenesis as a consequence of the embryo's environment. They are alike, however, in that in both (i) the primary sex determiner operates as a trigger that initiates the cascade of events that shape sex differences, and (ii) hormones secreted by the embryonic gonad govern the subsequent differentiation and development of other components of sexuality (Crews and Bull, 1987; Wilson et al., 1981).
A major variant of environmental sex determination is temperature-dependent sex determination (TSD). TSD was originally discovered in the African rainbow lizard (Agama agama) in 1966 by Madeline Charnier. Extensive research has revealed that in many oviparous reptiles, the temperature during the middle-third of embryogenesis is the critical cue determining the gonadal sex of the hatchling (Bull, 1980; Janzen and Paukstis, 1991). In TSD, sex determination operates as a switch mechanism that appears to be absolute; no individuals of ambiguous or intermediate sex are produced (Bull, 1985a and b, 1987a and b; Wibbels et al., 1991a). Because gonadal sex is determined after fertilization and egg-Laying in TSD, the primary sex ratio can be manipulated simply by varying incubation conditions.
Administration of exogenous estrogen to eggs of reptiles with TSD will override the effects of a male-producing temperature, resulting in a female hatchling (reviewed by Raynaud and Pieau, 1985; see also Bull et al., 1988; Crews et al., 1989, 1991; Gutzke and Bull, 1986). Studies at the University of Texas at Austin as well as other institutions have shown that the estrogen acts within a particular time window that corresponds with the window of temperature-sensitivity (Gutzke and Chyimi, 1988; Wibbels et al., 1991b). Further, estrogen treatments mimic the effects of female-producing temperature, resulting in the production of all normal-appearing female hatchlings (Crews et al., 1991). Studies with the leopard gecko lizard (Eublepharis macularius), a species with TSD indicate that these estrogen-reversed individuals are functionally female as adults (D. Crews, unpublished data).
Recently, it has been found that if eggs of red-eared sliders (Trachemys scripta) are incubated at a temperature that normally results in a 50:50 sex ratio, administration of dihydrotestosterone, a natural nonaromatizable androgen, causes male development (Wibbels et al., 1992). That the androgen must be nonaromatizable is important as administration of testosterone, a natural androgen that is converted by the enzyme aromatase to estradiol, causes female development (Bull et al., 1988; Crews et al., 1989; Gutzke and Bull, 1986).
The prior art shows that like temperature, steroid hormones can guarantee sexual development in TSD reptiles. Further, studies show clearly that male and female development in such species are two interlocking cascades that, depending on the steroid hormone used, can be independently activated.
The method of hormone treating eggs with exogenous sex steroid hormones by dipping or by injection has a long history. It is widely assumed that in species with sex chromosomes that steroid hormones play no role in sex determination and application of exogenous steroid hormones do not alter the primary sex ratio. This is the case in mammals (George and Wilson, 1988). In poultry (e.g., chickens, turkeys, etc.), which also have sex chromosomes, estrogen applied to the embryo will feminize the urogenital system of genetic male hatchlings, but they will revert to male plumage and gonads on attaining puberty; this includes the so-called "Selzer" method (van Tienhoven, 1957; Pincus and Hopkins, 1958). Administration of an aromatase inhibitor causes genetic female chickens to develop as phenotypic males (Elbrecht and Smith, 1992); whether these individuals can reproduce as males remains to be demonstrated.
In modern evolutionary biology, the birds are considered "reptiles" in that they evolved from reptile-like ancestors (see FIG. 2). Ratite birds (e.g. ostrich, emu, rhea, kiwi, and cassowary) represent the most primitive of extant birds. On the basis of skull morphology they are more similar to crocodilians than they are to other birds. Further, despite efforts using karyotyping and molecular genetic techniques for detecting sequence repeats, there is no evidence in the prior art to indicate that the ratites possess distinct sex chromosome as do other "higher" birds. The present invention illustrates that sex determination in the ratite birds, such as ostriches, is similar to that of reptiles with TSD in that sex can be manipulated via treatments of exogenous steroid hormones or their agonists.