Sex Determination
In the 1940’s, the French embryologist Alfred Jost observed that when the undifferentiated gonads were removed from a male rabbit fetus before male development had begun, it developed as a female. In 1959, chromosomal analysis of two disorders in man, Turner syndrome and Klinefelter syndrome, yielded the first evidence that genetic factors on the Y chromosomes of mammals are important in determining male sex. A specific gene on the mammalian Y chromosome (SRY, sex-related Y) induces male sex development during embryogenesis (sex determination).

• Determination of male phenotype by the Y chromosome
  
  Individuals with Turner syndrome have only one X chromosome (no Y chromosome) and a female phenotype, although incompletely developed and usually accompanied bymalformations. Individuals with Klinefelter syndrome have two X chromosomes, a Y chromosome, and a male phenotype, although also incompletely developed (p. 402).
  
  
• Sex-determining region SRY on the Y chromosome
  
  The relevant region in man lies in the distal short arm of the Y chromosome at Yp11.32. The short arm and the proximal half of the long arm of the Y chromosome have been divided into seven intervals (2). The most distal region of the short arm is the pseudoautosomal region 1 (PAR 1). This region is homologous to the distal segment of the short arm of the X chromosome. Homologous pairing occurs here with crossingover during meiosis. The physical map of the pseudoautosomal region and the proximal half of interval 1 (1A1–1B) span somewhat more than 2500 kb in man (3). Intervals 2–7 contain no genes for male sex determination. The crucial portion of the Y chromosome for male sex determination in man is about 35 kb (in the mouse about 14 kb) of a region designated Sry (sex-related Y chromosomal region) in the interval 1A1 proximal to the pseudoautosomal region (1). (After U.Wolf et al., 1992).
  
  
• Male development of an XX mouse transgenic for the Sry gene
  
  Clinical observations and experimental evidence indicate that the presence of SRY induces male development, irrespective of the presence of the remainder of the Y chromosome. A chromosomally female transgenic mouse (XX) shows normal male development after the 14 kb DNA fragment carrying the Sry region of a mouse Y chromosome is implanted into its blastocyst. (Figure from Koopman et al., 1991).
  
  
• Sry expression during embryonic gonadal development of the mouse
  
  During embryonic development of an XY mouse, Sry is expressed only between days 10.5 and 12.5. The subsequent events leading to male development are initiated during this short time of expression. (Figure from Koopman and Gubbay, 1991).
  
  
• References
  
  Cameron, F., Sinclair A.H.:Mutations in SRY and
  SOX9: testis-determining genes. Hum.
  Mutat. 9:388–395, 1997.
  Goodfellow, P.N., Camerino, G.: DAX-1, an “antitestis”
  gene. Cell Mol. Life Sci. 55:857–863,
  1999.
  Hargraeve, T.B.: Understanding the Y chromosome.
  Lancet 354:1746–1747, 1999.
  Koopman, P., Gubbay J.: The biology of Sry. Seminars
  Develop. Biol. 2:259–264, 1991.
  Koopman, P., et al.: Male development of chromosomally
  female mice transgenic for Sry.
  Nature 351:117–121, 1991.
  McElreavey, K., Fellous, M.: Sex determination
  and the Y chromosome. Am. J. Med. Genet.
  (Semin. Med. Genet.) 89:176–185, 1999.
  Roberts, L.M., Shen, J., Ingraham, H.A.: New solutions
  to an ancient riddle: Defining the
  differences between Adam and Eve. Am. J.
  Hum. Genet. 65:933–942, 1999.
  Swain, A., Lovell-Badge, R.: Mammalian sex determination:
  amolecular drama. Genes Dev.
  13:755–767, 1999.
  Wolf,U., Schempp,W., Scherer,G.: Molecular biology
  of the human Y chromosome. Rev.
  Physiol. Biochem. Pharmacol. 121:148–213,
  1992.
  
  

• Indifferent anlagen of sex differentiation
  
  The gonads (1), the efferent ducts (mesonephric and paramesonephric) (2), and the external genitalia (3) all develop from an indifferent stage. At about the end of the sixth week of pregnancy in humans, after the primordial germ cells of the embryo have migrated into the initially undifferentiated gonads, an inner portion (medulla) and an outer portion (cortex) of the gonads can be distinguished. When a normal Y chromosome is present, early embryonic testes develop at about the 10th week of pregnancy under the influence of a testis-determining factor (TDF). If a normal Y or TDF (SRY) is not present, ovaries develop. The wolffian ducts, the precursors of the male efferent ducts (vas deferens, seminal vesicles, and prostate), develop under the influence of testosterone, a male steroid hormone formed in the fetal testis. At the same time, the müllerian ducts—precursors of the fallopian tubes, the uterus, and the upper vagina—are suppressed by a hormone, the Müllerian Inhibition Factor (MIF; also known as anti-müllerian hormone, AMH). When testosterone is absent or ineffective, the wolffian ducts degenerate. The müllerian ducts develop under the influence of estradiol, a hormone produced by the fetal ovaries. The external genitalia (3) in humans do not develop until relatively late, starting in the 15th to 16thweek. Full development of male external genitalia depends on a derivative of male-inducing testosterone, 5-dihydrotestosterone, a metabolite of testosterone produced by the enzymatic action of 5!-reductase.
  
  
• Sequence of events in sex differentiation
  
  Sex differentiation proceeds in a cascadelike manner, with a series of temporally regulated successive steps at different levels of differentiation. After the primordial germ cells migrate into the undifferentiated gonads, early embryonic testes develop under the influence of testis-determining factor (TDF) if a Y chromosome is present. TDF is identical with the Yspecific sequences of the SRY region (see p. 386). During normal male differentiation, the further development of the müllerian ducts is suppressed by the müllerian inhibitor factor. Testosterone can exert its effect only in the presence of an appropriate intracellular receptor (androgen receptor TFM, see p. 390). When a Y chromosome is not present or when the SRY region is missing or altered by mutation, testes are not formed. In this case the wolffian ducts cease to develop. In the absence of testes, ovaries develop from the undifferentiated gonads; the wolffian ducts degenerate; and the müllerian ducts differentiate into uterine tubes, uterus, and the upper vagina. Testosterone also has an effect on the central nervous system (“brain imprinting”). It is assumed that this is required for the psychosexual orientation apparent later in life. When testosterone is absent or ineffective due to a receptor defect, gender orientation is female. In the majority of genetically determined disorders of sexual differentiation, gonadal and genital sex do not correspond (pseudohermaphroditism). In true hermaphroditism, where the gonads consist of both testicular and ovarian tissues, male and female structures exist side by side.
  
  
• References
  
  Arango, N.A., Lovell-Badge, R., Behringer, R.R.:
  Targeted mutagenesis of the endogenous
  mouse Mis gene promoter: in vivo definition
  of genetic pathways of vertebrate
  sexual development. Cell 99:409–419,
  1999.
  Ferguson-Smith, M.A., Goodfellow, P.N.: SRY
  and primary sex-reversal syndromes, pp.
  739–749, In: C.R. Scriver, et al., eds., The
  Metabolic and Molecular Bases of Inherited
  Disease. 7th ed. McGraw-Hill, New York,
  1995.
  Wilson, J.D., Griffin, J.E.: Disorders of sexual
  differentiation, pp. 2119–2131. In: A.S.
  Fauci, et al., eds., Harrison’s Principles and
  Practice of Internal Medicine. 14th ed..
  McGraw-Hill, New York, 1998.

• Male-determining region SRY on the Y chromosome
  
  Normally, the male-determining Y-specific DNA sequences (SRY) remain on the Y chromosome during the homologous pairing and crossingover during meiosis. However, since the maledetermining region SRY is located very close to the pseudoautosomal region (PAR), crossingover in the PAR border region may result in a transfer of the SRY region to the X chromosome. This results in a male individual with an XX karyotype (XX male). Conversely, if the SRY region is missing from a Y chromosome, a female phenotype with XY chromosomes (XY female) results.
  
  
• Point mutations in the SRY gene
  
  The human SRY gene has a single exon and encodes a 204-amino-acid protein from a 1.1 kb transcript. The middle section of the SRY protein consists of 79 highly conserved amino acids with DNA-bending and DNA-binding capability, the HMG box (high mobility group protein). Complete or partial gonadal dysgenesis results from point mutations and deletions in the SRY gene, in particular the HMG box. (Figure adapted fromWolf et al., 1992; for an update of mutations see McElreavey and Fellous, 1999). Sex reversal also results from mutations in the SOX9 gene on chromosome 17 at q24 in campomelic dysplasia. (Foster et al., 1994; Wagner et al., 1994).
  
  
• Androgen receptor
  
  The fetal testis produces testosterone, the hormone that induces male sexual differentiation. Testosterone is taken up by cells of the target tissues (wolffian ducts and urogenital sinus) (1). In the urogenital sinus, testosterone is converted into dihydrotestosterone (DHT) by the enzyme 5!-reductase. Both testosterone and dihydrotestosterone bind to an intracellular receptor (androgen receptor). The activated hormone– receptor complex (TR* or DR*) acts as a transcription factor for genes that regulate the differentiation of thewolffian ducts and the urogenital sinus. Thus, normal male fetal development is dependent on normal biosynthesis of testosterone and normal receptors. Androgen receptor mutations lead to disorders of sexual development (2) with X-chromosomal inherited complete or incomplete androgen resistance (testicular feminization, TFM).
  
  
• Classification of genetically determined disorders of sexual development
  
  1. Defects of sex determination due to mutation or structural aberration of the SRY region
  on the Y chromosome (e.g., XY gonadal dysgenesis, XX males, and others)
  
  2. Defects of androgen biosynthesis (e.g., adrenogenital syndrome due to 21-hydroxylase
  deficiency, see p. 392)
  
  3. Defects of androgen receptors (testicular feminization)
  
  4. Defects of the müllerian inhibition substance (so-called hernia uteri syndrome)
  
  5. XO/XY gonadal dysgenesis
  
  
• References
  
  Foster, J.W., et al.: Campomelic dysplasia and
  autosomal sex reversal caused bymutations
  in an SRY-related gene. Nature 372: 525–
  530, 1994.
  Goodfellow, P.N., Camerino, G.: DAX-1, an “antitestis”
  gene. Cell Mol. Life Sci. 55:857–863,
  1999.
  Gottlieb, B., et al.: Androgen insensitivity. Am. J.
  Med. Genet. (Semin. Med. Genet.) 89:210–
  217, 1999.
  McElreavey, K., Fellous, M.: Sex determination
  and the Y chromosome. Am. J. Med. Genet.
  89:176–185, 1999.
  Wagner, T., et al.: Autosomal sex reversal and
  campomelic dysplasia are caused by mutations
  in and around the SRY-related
  SOX9. Cell 79:1111–1120, 1994.
  Wilson, J.D., Griffin, J.E.: Disorders of sexual
  differentiation, pp. 2119–2131. In: A.S.
  Fauci, et al., eds., Harrison’s Principles and
  Practice of Internal Medicine. 14th ed.,
  McGraw-Hill, New York, 1998.

• Clinical phenotype and genetics
  
  Girls are born with ambiguous or virilized genitalia (1). The adrenal cortex is enlarged (2). Increased production of androgenic metabolites causes masculinization. The cortisol deficiency (3) leads to life-threatening crises due to loss of sodium chloride (salt-wasting) that require prompt treatment. AGS is an autosomal recessive heritable disorder (4). Untreated girls develop amale physical appearance (5). In boys, the early signs are limited to salt-wasting. Initially, skeletalmaturation is accelerated and the children are tall for their age; however, they stop growing prematurely and eventually are too short. Besides the classic form of the disorder with a frequency of 1:5000, there are other forms with less pronounced masculinization due to different mutations.
  
  
• Biochemical defect
  
  The enzymatic conversion of progesterone to deoxycortisol (DOC) by hydroxylation at position 21 (steroid 21-hydroxylase) is decreased. As a result, the concentration of 17-hydroxyprogesterone is increased.
  
  
• Gene locus and gene structure
  
  21-Hydroxylase is encoded by the CYP21 gene (formerly called CYP21B and 21-OHB), a member of the cytochrome P450 oxidase gene family. This gene is located within the class III genes of the major histocompatibility complex on the short arm of human chromosome 6. It is part of a tandem paired arrangement of three other genes: active C4A and C4B genes and a 96–98% homologous inactive CYP21P gene, a pseudogene due to intragenic deletions resulting in stop codons (formerly called CYP21A or 21-OHA). These genes originated from a duplication event in evolution. The CYP21 (21-OHB) gene consists of 10 exons spanning almost 6 kb of genomic DNA (the actual distance of 30 kb to the C4 A and C4 B genes is not shown to scale).
  
  
• Molecular genetic analysis
  
  Point mutations, deletions, and duplications occur in the CYP21 gene. The deletions and duplications result from misalignment of the homologous chromatids during meiosis and unequal crossing-over. Deletions occur in about 20–25% of patientswith classic 21-hydroxylase deficiency. Duplications have no clinical consequences. Deletions and duplications can be easily detected by Southern blot analysis. The most frequent type of deletion is loss of a 30 kb region including the 3' part of the CYP21P pseudogene, the entire C4B gene, and the 5' part of the CYP21 gene. The resulting fusion gene of CYP21P and CYP21 carries a TaqI restriction site in the 5' region of CYP21P that is not present in the CYP21 gene. Therefore, the fusion gene has a characteristic 3.2 kb TaqI fragment. This distinguishes the rearrangement from the normal CYP21 gene, which has a characteristic 3.7 kb fragment. In the example shown, the CYP21 gene (21-OHB) is represented by a 3.7 kb DNA fragment, the pseudogene CYP21P (21-OHA) by a 3.2 kb fragment after TaqI digestion (1). Thus, the normal pattern is a 3.7 kb and a 3.2 kb fragment (2). Homozygous deletion of either of the genes may be apparent by lack of either of the two fragments (3, 4). A heterozygous deletion shows reduced intensity (5) and a duplication shows increased intensity (6). (Figure adapted from New et al., 1989). Another common mechanism for the origin of mutation in the CYP21 gene is gene conversion. This involves nonreciprocal exchange between the closely linked CYP21 and CYP21P genes, which results in transfer of mutations from the pseudogene CYP21P to CYP21.
  
  
• References
  
  New, M.I., et al.: The adrenal hyperplasias, pp. 1881–1917. In: C.R. Scriver, et al., eds., The Metabolic Basis of Inherited Disease. 6th ed. McGraw-Hill, New York, 1989. Wilson, R.C., New, M.I.: Congenital adrenal hyperplasia, pp. 481–493. In: J.L. Jameson, ed., Principles of Molecular Medicine. Humana Press, Totowa, New Jersey, 1998.