Allen S. Goldman,     Division of Experimental Pathology, Children’s Hospital of Philadelphia; and the Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. 19104

Publisher Summary

The inborn program of sexual differentiation in the mammalian fetus is female unless there is a testis-secreting hormone—testosterone—that transforms the originally bisexual fetus or neonate into a male. This chapter explores several of the possible parameters of sexual differentiation that may be programmed in utero or in neonatal life. The chapter explains how the natural and experimental testosterone-depriving agents may elucidate the programming. It has been observed that cyproterone acetate—a progestin with potent antiandrogenic action, which blocks testosterone and dihydrotestosterone uptake in target organs—prevents normal masculine differentiation as well as all androgen-dependent anatomic and hypothalamic differentiation. The development of highly specific and potent inhibitors of testosterone biosynthetic enzymes and the use of antibodies to testosterone and antibodies to LH: FSH have provided selective experimental agents for testosterone deprivation. These agents produce a reversible chemical castration by ablation of testosterone at the level of its synthesis, circulation, or uptake. The use of the experimental and genetic testosterone-depriving agents has substantiated the hypothesis that testosterone is the organizer of androgen-dependent differentiation.

Introduction

The inborn program of sexual differentiation in the mammalian fetus is female. The program is female unless there is a testis-secreting hormone (i. e., testosterone) which transforms the originally bisexual fetus or neonate into a male. The history of the experimentation from which this fundamental statement is derived traces the exciting application of the three basic techniques of classical endocrinology to the study of the sexual development of the fetus and neonate. The first technique of determining responses to exogenous gonadal hormones was applied with conflicting results in the 1930’s soon after the first chemical syntheses of these hormones. As may be expected, testosterone virilized female offspring, and estradiol-17β feminized male fetuses. However, the paradoxic effects of estradiol-17β, that is, virilization of females and production of adrenal enlargement in both sexes, allowed no definitive conclusions about the roles of these hormones in normal sexual differentiation. Moreover, in 1942, Counter and Jost warned against the use in pregnant women of a third class of gonadal hormones – derivatives of progesterone, i. e., progestational agents – on the basis of paradoxic effects of these agents [7]. They observed that ethisterone, as an example of a progestin, had three effects on the pregnant rabbit: a progestational action with maintenance of gestation, a masculinizing effect on the female fetus, and a slight feminizing effect on the male fetus.

Hypothalamic Sex

Pfeiffer, in 1936, utilizing the remaining classical techniques of castration and hormone replacement in the neonatal rat, provided the first definitive evidence that male sex hormones, secreted in the first few postnatal days of life, transform the indifferent hypothalamus into the masculine type, i. e., a tonic secretion of gonadotrophins after puberty (Fig. 1) [35]. In the absence of a testis, either sex develops the female pattern of a cyclic secretion of gonadotrophins after puberty. Testosterone replacement of the neonatal castrate restores the masculine pattern of differentiation.

Anatomic Sex

Jost, by his experimental embryological investigations in the late 1940’s, introduced fetal castration with testosterone replacement and demonstrated that the inborn sexual programing of mammalian anatomic sex – i.e., differentiation of the external genitalia, mammary glands, and Wolffian duct structures – is also feminine unless there is a functioning testis (Fig. 2) [29]. He showed that removal of the fetal gonad leads to feminization of the external genitalia, expression of mammary gland development, and the absence of Wolffian duct development in either sex. Testosterone corrects the defect in all androgen-dependent masculine development produced by castration of male fetuses with the exception of Muellerian duct regression. This appears to be under the control of Sertoli cell production of some kind of protein Muellerian inhibitor [27, 28]. Impairment of fetal testicular function (produced by fetal decapitation as a form of hypophysectomy) leads in the rabbit to hypospadias as an external sign of incomplete masculine differentiation [29]. Similar studies of fetal decapitation in the rat gave inconclusive results.

Sexual Programing of Human Anatomic Sex

The suggestion that the fetal testis converts the feminine inborn programing to masculine differentiation in the human was first provided by the observation of Bongiovanni and associates. They noted hypospadias in boys with a rare form of congenital adrenal hyperplasia due to a genetic deficiency of 3β-hydroxy-Δ5-steroid oxidoreductase [3], and suggested that a deficiency of this enzyme in the testis provides a genetic castrate in utero by virtue of the fact that testosterone biosynthesis is blocked at the level of the weak Δ5,3β-hydroxysteroid androgens. Bongiovanni noted the similarity of this human hypospadic boy to the hypospadic fetal decapitated male rabbit and suggested that the etiology of the human disease is due to deficient testosterone production. More recently, two other rare forms of congenital adrenal hyperplasia due to genetic defects in 17α-hydroxylase and cholesterol desmolase [3] – as well as the primarily testicular enzyme defect, 17-ketoreductase [36] – also result in genetic testosterone deprivation with feminine anatomic sex in boys.

Testicular Feminization

One other human disease, testicular feminization, is an example of genetic castration by virtue of an inherited defect of androgen unresponsiveness. The nature of the genetic defect in the animal counterpart, the male rat pseudohermaphrodite (Stanley-Gumbreck) has been convincingly demonstrated to be a deficiency of androgen receptor proteins in the target organs [2]. This animal is a prototype of feminine differentiation of a chromosomal male with genetic castration in utero (Fig. 1).

Biochemical Sex

In the 1960’s, the work of Kraulis and Clayton [31] and DeMoor and Denef [8] introduced the biochemical level of organization to the definition of sex differentiation (Fig. 3). They showed that the mechanisms which control qualitatively and quantitatively different sex-dependent patterns of enzyme levels in the liver are irreversibly determined by the testis of the male in the first few days of life. Male rats castrated in the neonatal period develop, after puberty, a female type of hepatic metabolism [8, 31]. Testosterone replacement of the neonate irreversibly restores the male pattern but, given after puberty, produces only a transient male pattern. Recently, Gustafsson et al. have studied these imprinting mechanisms in more detail and have found that the steroid-metabolizing enzymes in rat liver can be classified in different categories according to their mode of sexual regulation [10]. Gustafsson et al. have also shown that the androgen responsiveness of the rat liver is determined by neonatal imprinting of testicular androgens [26]. The male rat pseudohermaphrodite has a female pattern of 5α-reduction which is reversed by testosterone in the adult [9, 17, 25].