The first step in sex determination and differentiation is the establishment of genetic sex (4). When fertilization occurs between an oocyte (carrying an X chromosome) and a sperm (carrying either an X or a Y chromosome) the result is a zygote that is genetically female (46,XX) or male (46,XY).
The formation of bipotential gonadal ridges, Müllerian and Wolffian duct structures common to both 46,XX and 46,XY embryos is evident by week 5 of gestation (5). Multiple transcription factors are involved in this phenomenon including LIM-1, EMX-2, PAX-2, WT-1, SF-1 and DAX-1 as well as the growth factors DMRT1, ATRX and insulin receptors (4-8). In mice, the development and maintenance of bipotential gonadal tissue requires expression of the genes M33, Emx2, Dhx9, Pod1 and Dmrt1, as well as Sf1 and Wt1 (5, 8).
The differentiation of ovaries or testes from the bipotential gonadal ridge tissue is the next step in normal sex differentiation. In humans, differentiation of the testes and ovaries is initiated by 6 weeks and fully achieved by 13 to 14 weeks of fetal development. Testicular differentiation is controlled by a number of time- and dosage-sensitive genes that encode for transcription factors (4). These include factors that are positive for the formation of testes: (WT-1, SOX-9 and the sex determining region of the Y chromosome (SRY)) and others that inhibit testicular differentiation: (DAX-1, SOX-3 and WNT-4 (1, 9, 10). Among 46,XY individuals affected by disorders of testicular differentiation and development, most of the identified mutations have been observed in SF-1, SOX-9 or SRY (11).
Ovarian differentiation is less well understood and was previously believed to be the default state of gonadal differentiation. Recent research shows that ovarian development is an active process that occurs in the absence of SRY coupled with the expression of additional genes including FOXL-2, WNT-4 and possibly DAX-1 (12, 13).
The fetal production of testicular androgens and Müllerian inhibiting substance (MIS) constitute the next step of sex differentiation and development. In males, androgens are necessary to both masculinize the external genitalia and to support the maintenance of the Wolffian ducts, while MIS suppresses Müllerian duct development. The biosynthesis of testosterone by the Leydig cells requires the activity of a series of enzymes (14). Expression of androgenic effects by target tissues such as Wolffian duct structures requires the binding of testosterone to androgen receptors (15).
In females, fetal ovaries do not produce MIS until after the fetus is committed to developing along female lines (14); hence, development and maintenance of the Müllerian duct structures are permitted. Furthermore, because the fetal ovaries do not secrete androgens, the Wolffian ducts become atrophic and the external genitalia continue to appear female in newborn girls.
In 46,XY fetuses the female-appearing external genitalia start to masculinize at 8-9 weeks of gestation when the potent androgen dihydrotestosterone (DHT) is present. DHT is required for fusion of the urethral and labioscrotal folds, lengthening of the genital tubercle, and regression of the urogenital sinus (16). Steroid 5α-reductase-2 is the enzyme required to convert testosterone that is produced by the fetal Leydig cells to DHT. Steroid 5α-reductase-2 is located in androgen target cells of the developing external genitalia, but not in the testes. Complete masculinization of the external genitalia by DHT is accomplished by week 14 of gestation. In the absence of either testosterone secretion or 5α-reductase-2, the fetal external genitalia continue to develop along female lines. Specifically, without DHT the labioscrotal and urethral folds form the labia majora and minora, respectively. The genital tubercle develops into a clitoris and the urogenital sinus gives rise to the urethral opening and anterior portion of the vagina (17). The posterior portion of the vagina develops from the Müllerian ducts in the absence of MIS.