Indian J Pediatr 1992; 59 : 467-473 F
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Embryology and Classification of lntersex States Nathalie Josso
Unite de Redterches stir L' Endocrinologie dtt Developpement, INSERa~I, I-t6pital des Enfants-Malades, Paris, France From the time of fertilization to birth, the mammalian conccptus develops from an egg to an individual characterized by a defined phcnotypic sex, either male or female. Sex differentiation can be defined as the "cascade" leading from genotypic to phenotypic sex. It is achieved before birth in all eutherian mammals. The pioneering experiments of Jost ~'2 have clearly illustrated the assymetrical basis of sex differentiation. Constitutively, the mammalian embryo is programmed to develop as a female, and male-determining agents are required to impose masculine characterislics on the body. The hinge between genetic and hormonal agents of male sex differentiation is located at the level of the fetal gonad: diverted towards testicular differentiation by Y-located genes, it produces in turn hormones which trigger male development of the internal and external reproductive tract. TESTICULAR DIFFERENTIATION In the mammalian fetus, the gonadal priReprint requests : Dr. Nathalie Josso, Unite de Recherches sur L' Endocrinologie du Dcveloppment, INSERM, Hopital des EnfantsMalades, Paris, France
mordium is represented by the gonadal ridge, a thickening of the coelomic epithelium covering tee anterior surface of the mesonephros, which is progressively colo, nized by primordial germ ceils travelling from the stalk of the allantois through the mesentery and the wall of the fetal gut ~. The first recognizable event of testicular differentiation is the development of a new cell type, the primordial Sertoli cells, which soon aggregate to form seminiferous tubules in which germ cells become enclosed46. In contrast to extratesticular germ cells, which, like female ones, enter the prophase of the first meiotic division early in fetal life7, spermatogonia enclosed in testicular tubules do not undergo meiosis until the beginning of puberty. In the human fetus, the testis can be recognized as early as 7 weeks after fertilization. Fetal Sertoli cells art large, clear cells, with abundant cytoplasm containing vesicles of rough endoplasmic reticulum. In these vesicles is: stored s, prior to secretion, a glycoprotein, anti-Miillerian hormone (AMIt), which is responsible for the inhibition of the development of the Miillerian ducts in male fetuses x,2, and which may also play a role in gonadal differentiation, as will be discussed later. Scrtoli cells continue to produce AMH throughout the whole of
This article is based on the presentation in the "International Workshop on Recent Advances in Neonatal Surgery and Intcrsex Disorders" hcld at All India Institute of Medical Sciences, New Delhi from March 1-4, 1989.It was accepted for publication in 1991.
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THE INDIAN JOURNAL OF PEDIATRICS
gestation, and in the immediate postnatal period (reviewed in relY). Leydig cells differentiate somewhat later, at 8 weeks of gcstation in the human fetus. Their number increases dramatically until 14 to 16 weeks, and then Lcydig cells degenerate, and just prior to birth, very few are still visible in the interstitial tissue. Fetal Leydig cells, stimulated by chorionic gonadotropin, produce testosterone, which is responsible for the male dcvclopment of Wolffian derivatives, urogenital sinus and external genital organs. Agents of Teslieular Differentiation
Vol. 59, No. 4
hunt for Z F Y / Z F X related sequences had led to the disquieting mapping of homologous sequences of autosomal DNA of marsupials t4 implying that ZFY is not the testisdetermining gene in this therian subclass, which divcrged from placental mammals 45 million ycars ago. SRY does not act directly upon gonadal sex differentiation. The structure of the encoded protein is typical of one with DNAbinding affinity. Since testicular sex differentiation is a complex process, inw)lving also autosomal genes ~5, the protein encoded by SRY could play a regulatory role by activating or inhibiting the transcription of other genes. Proteins encoded by such "downstream genes" should exert a testis-determining activity per se. In this regard, it may be interesting to note that anti-Miillerian hormone (AMH) has recently been shown to mascullnize the structure and function of fetal ovaries in vitro 16,x7and is therefor.-, a candidate for SRY regulation. Fetal ovaries exposed to AMH in vitro divert their hormonal output from estrogen to testosterone production ~8.
What induces the fetal gonad to develop into a testis? The critical role of the Y chromosome in mammals has been recognized for a number cf years: regardless of the number of X chromosomes in their karyotype, cmbryos carrying a Y chromosome develop as males. This rule, howevcr, is not absolute : some XX individuals develop into males ~~ in contrast, chromosomal males with a female phenotype have been identified n. The genetic analysis of the DNA of these patients, pursued by a posse of investigators has becn crowned by the isolation FETAL TESTICULAR HORMONES of a sex determining gene, SRY ~2, coding for a DNA-binding protein, often called tesAs reviewed above, testis-determining tis-determining factor (TdF; referred to as genes are responsible for diverting an emTdy in the mouse). The SRY probe detects bryonic gonad from a constitutive female male-specific sequences in most-but not all- pathway towards testicular differentiation. XX males, but does not hybridize to Testis-determining genes are not involved DNA of XY females, leading to the conclu- in somatic sex differentiation, which is insion that translocation or deletion of this duced by fetal testicular hormones, regardDNA sequence is responsible for the appar- less of genotype. The classical experiments ent discrepancy between sex genotype and of Jost 2s have clearly demonstrated that the phenotype. SRY is highly conserved, and fetal ovaries play no active part in somatic shows similarities with products of yeast sex differentiation: agonadal fetuses differgenes involvcd in mating. A previous candi- entiate normally as females. Thus, the date for TDF, a zinc-finger transcription assymetrical nature of sex differentiation is unit (ZFY) had been put forward ~3but the reiterated: the testes must impose mascu-
JOSSO : E M B R Y O L O G Y A N D CLASSII:ICATION OF I N q ~ R S E X STATES
linity upon a body which would otherwise develop along female lines. Failure of the testes to carry out their mission-through incapacity to synthesize the appropriate hormones, or through end-organ resistance to these hormones-leads to male pseudohermaphroditism. As demonstrated by Jost 2, two discrete hormones are synthesized by the fetal testis : the Miillerian inhibitor (AMH) and testosterone. Anti-Miillerian Hormone
Anti-Miillerian hormone (AMH) is a 145,000 dimer containing 13.5% carbohydrate synthesized by immature Sertoli cells s and also by adult granulosa cells (see ref9 for review). Bovine testicular AMH has bccn purified to homogeneity by immunochromatography on a monoclonal antibody19, and the gencs coding for bovine and human AMH 2~have been cloned. The gene has been localized to the tip of the short arm of human chromosome 192I. The C-terminal part of the molecule shares a # 30% homology with the beta subunit of bovine inhlbin, with transforming growth factor beta 2~ whose genc has also been mapped to human chromosomQ 19, with the product of the decapentaplegic complex of Drosophila 22 and the Vgl gene of Xenopus 23. Proteolytic processing of recombinant AMH (MIS) produces a transforming growth factor-beta-like fragment ~. AMH is responsible for the regression of Miillerian derivatives in the male fetus 1'2 and has also been shown to masculinize fetal ovaries (see above). According to Hutson and Donahoe z~ AMH could also promote testicular descent, but no experimental evidence for this hypothesis has been provided. To the contrary, Fentener van Vlissingen et a126 have shown that AMH does not stimulate the growth of guber-
469
naculum cells in vitro, whereas a fetal testicular extract is active in this system. Partially purified bovine AMH has been reported to exert art antiproliferative effect on malignant cell lines of gynecological origin 27 but this has not been confirmed using an hormone purified to homogeneity~. Human recombinant AMH has no anti-cancer activity 29. Testosterone
Testosterone is produced by fetal Leydig cells from the time of their differentiation at 8 weeks of age. Initiation of testosterone secretion requires no hormonal sfimulation, but continued production requires the presence of gonadotropin 3~ which explains why the serum levels of testosterone and hCG are well correlated in the human fetus31. Testosterone exerts its biological effect through binding to an X-linked receptor molecule3z, which has a much greater affinity for dihydrotestosterone (DHT) than for testosterone itself. Therefore, in tissues containing alpha-reductase, the enzyme metabolizing testosterone to DHT, DHT is in fact the active androgen. The gene coding for the androgen receptor has recently been cloned from a flow-sorted human chromosome library, using as probe a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors 33. The deduced aminoacid sequence for the DNA-binding domain of the androgen receptor eDNA closely resembles that of the progesterone, mineralocorticoid and glucocorticoid receptor, and contains cysteine residues which could be involved in "finger" formation. NORMAL AND ABNORMAL MALE SOMATIC SEX DIFFERENTIATION Normal male somatic sex differentiation
470
THE INDIAN JOURNAL OF PEDIATRICS
is characterized by regression of Miallerian derivatives, maintenance and differentiation of the Wolffian ducts into vasa deferentia, epididymes and seminal vesicles, and the virilization of the urogenital sinus and external genitalia. Miillerian Duct Regression
AMH-mcdiated Miillerian duct regression begins at 8 weeks in the human fetus, and is complete at approximately 9 weeks34. Relatively high levels of AMH have been detected in the serum of fetal ruminants and human males up to puberty (reviewed in reference 9). In the human fetus, AMH could act by local diffusion from the testis to the adjacent Miillerian duct : in alternating true hermaphroditism, and in mixed gonadal dysgenesis, the unilateral testis has no inh!bitory effect upon the contralateral Miillefiah duct. Being a polypeptide hormone, AMH probably affects its target organ through binding to a membrane receptor molecule, but this hypothesis has not yet been borne out by experimcntat proof. Abnormal persistence of Mtillerian derivatives has been described in otherwise normal males35. The molecular basis of this condition is heterogeneous : in some, but not all, patients, immature testicular tissue expresses AMH normally, suggesting that the persistent Miillerian duct syndrome, a genetically transmitted defect, could be due to mutations affecting the biosynthesis of either the hormone itself, or of its putative receptor. In other instances, persistence of MiJllerian derivalives is associated with impairment of testosterone-dependent steps of sex differentiation, and is a consequence of testicular dysgenesis36.
Vol. 59, No. 4
The Woiffian Ducts
Wolffian ducts, initially the excretory ducts of the primitive mesonephros, become testosterone dependent later, when they become part of the male genital system. In humans, Wolffian ducts depend for their differentiation essentially upon the amount of testosterone they can take up locally (33). Testosterone itself, and not DHT, is the active androgen, since fetal Wolffian ducts contain no 5-alpha-reductase37. Urogenital Sinus and External Genitalia
Virilization of these structures is initiated at 10 weeks in the human fetus. It is characterized by the inhibition of the development of the vaginal plate, the development of prostatic buds, the growth of the genital tubercle and the closure of the urogenital folds. In these tissues, testosterone is reduced to DHT by 5-alpha reductase, binds to intracellular androgen receptors which then activate the transcription of virilizing genes. Incomplete external virilization can be due to various causes. Defects in testosterone synthesis by the fetal testis may be caused by Leydig cell agencsis38, generalized testicular dysgenesis3~, or may arise from congenital defects of steroidogenesis. Peripheral insensitivity to testosterone may be caused by defects of 5-alpha-reductase~9, or by androgen receptor defects4~ Recently, deletion of the steroid-binding domain of the human androgen receptor gene has been reported in a family with a receptor(-) form of the complete androgen insensitivity syndrome41. The cause of androgen insensitivity in patients cxhibiting quantitatively normal binding of androgen in cultured fibroblasts remains to be determined.
JOSSO : EMBRYOLOGY AND CLASSIFICATIONOF INTERSEX STATES Sex Differentiation of the Brain
Differentiation of neuroendocrine patterns and sex behaviour is regulated by testosterone during the neonatal period. In both humans 4z and rats 43, a peak of testicular activity is observed immediately after birth. As reviewed by MacLusky and Naftolin 44, neonatal and androgenization of rodents induccs a permanent change in hypothalamic function which leads to the suppression of the LH midcycle surge. Testosterone treatment also abolishes the sexual dimorphism of the spinal cord 45. In contrast to neuroendocrine sex differentiation, psychological sex differentiation in humans is thought to be acquired postnatally by sociologic imprinting on the developing personality 46. However, the determining influence of the sex of rearing has been questioned, and lmperato-McGinley et a147 believe that an initially female gender identity can evolve at puberty under the influence of rising androgen production by the testes. In conclusion, because disorders of sex differentiation do not seriously impair the somatic heallh of their victims, clinical studies provide "experiments of nature" to illustrate physiological hypotheses. This field invites the continued collaboration of physicians and basic scientists to unravel the last mysteries obscuring what Jost 48 has called a prolonged, uneasy and risky venture : becoming a male! REFERENCES i. Jost A. Recherches sur la diffdrenciation sexuelle de l'embroyn de lapin, lII. R61e des gonades foetales dans la diff6renciation sexuelle somatique. Arck Anat Mierosc Motphol E W 1947; 36:271-315. 2. Jost A. Problems of fetal endocrinology :
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the gonadal and hypophyseal hormones. Recent Progrtlonn Res 1953; 8 : 379-418. 3. McKay DG, Hertig AT, Adams EC et al. HistochemicaI observations on the germ cells of human embryos. Anat Ree 1953; 117 : 201-219. 4. Agelopoulou R, Magre S, Patsavoudi E e t al. Initial phases of the rat testis differentiation in vitro. J Embryol Exp Motphol 1984; 83 : 15-31. 5. Jost A. Donn6es pr61iminaires sur les stades initiaux de la diff6renci.ation du testicule chez le rat. Atvh Anat Microse Morphol Exp 1972; 61 : 415-437. 6. Magre S, Jost A. The initial phases of testicular organogenesis in the rat. An electron microscopy study. Arch Anat Mictvsc Morphol Exp 1980; 69:297-318. 7. Upadhyay S, Zamboni L. Ectopic germ cells - Natural model for the study of germ cell sexual differentiation. Proc Natl Acad Sci USA 1982; 79 : 6584-6588. 8. Tran D, Josso N. Localization of anti-Mfillerian hormone in the rough endoplasmic reticulum of the developing bovine Sertoli cell using immunocytochemistry with a monoclonal antibody. Endoclinology 1982; 111 : 1562-1567. 9. Josso N, Care RL, Picard JY ct al. AntiMfillerian hormone, the Jost factor. Recent Progrllotm Res 1992; 48 (in press). 10. De la Chapelle A, Koo GC, Wachtel SS. Recessive sex-determining genes in human XX male syndrome. Cell 1978; 15 : 837-842. 11. Disteche CM, Casanova M, Saal H c t al. Small deletions of the short arm of the Y chromosome in 46, XY females. Proc Natl Acad Sci USA 1986; 83 : 7841-7844. 12. Sinclair AH, Berta P, Palmer MS et al. A gene from the human sex-determining region encodes a protein with homology ~o a conserved DNA-binding motif. Nature 1990; 346 : 240-244. 13. Page DC, Mosher R, Simpson EM et al. The sex-determining region of the human Y chromosome encodes a finger protein. Cell 1987; 51 : 1091-1104.
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14. Sinclair AH, Foster JW, Spencer JA et al. Sequences homologous to ZFY, a candidate human sex-determining gene, are autosomal in marsupials. Nattoe 1988; 336 : 780-783. 15. Washburn LL, Etcher EM. Sex reversal in XY mice caused by dominant mutation on chromosome 17. Nantre 1983; 303 : 338-339. 16. Vigier B, Watrin F, Magre S et al. Purified bovine AMH induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to in vitro. Development 1987; 100 : 43-55. 17. di Clementc N, Ghaffari S, Popinsky RB et al. A quantitative and intcrspecific test for biological activity of anti-M/illerian hormone : the fetal ovary aromatase assay. Development 1992; 114:721-727. 18. Vigier B, Forest MG, Eychenne B et al. Anti-Mtitlerian hormone produces endocrine sex-reversal of fetal ovaries. Proc Natl Acad Sci USA t989; 86 : 3684-3688. 19. Picard JY, Josso N. Purification of testicular anti-Mfillerian hormone allowing direct visualization of the pure glycoprotein and determination of yield and purification factor. Mol Cell Endoctinol 1984; 34 : 23-29. 20. Cate RL, Mattaliano RJ, Hession C et al. Isolation of the bovine and tiuman genes for m/.illerian inhibiting substance and expression of the human gene in animal cells. Cells 1986; 45 : 685-698. 21. Cohen-Haguenauer O, Picard JY, Mattei MG et al. Mapping of the gene for antiM/illerian hormone to the short arm of human chromosome 19. Cytogenet Cell Genet 1987; 44 : 2-6. 22. Padgett RW, St-Johnston RD, Gelbart WM. A transcript from a Drosophila pattern gene predicts a protein homologous to ~he transforming growth factor-beta family. Nature 1987; 325 : 81-84. 23. Weeks DL, Melton DA. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell 1987; 51 : 861867.
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24. Pepinsky RB, Sinclair LK, Chow EP et al. Proteolytic processing of Mi~llerian inhibiting substance produces a transforming growth factor-a-like fragment. J Biol Chem 1988; 263 : 18961-18965. 25. Hutson JM, Donahoc PK. The hormonal control of testicular descent. Endocrine Rev 1986; 7 : 270-283. 26. Fentener van Vlissingen FM, Van Zoelen EJJ, Ursem PJF et al. In vitro model of the first phase of testicular descent : identification of a low molecular weight factor from fetal testis involved in proliferation of gubernaculum testis ceils and distinct from specified polypeptide growth factors and fetal gonadal hormones. Endocrinolo~r 1988; I23 : 2868-2877. 27. Donahoe PK, Budzik GP, Trelstad R et al. Mfillerian-inkibiting substance : an update. Recent ProgT'fIotm Res 1982; 38 : 279-326. 28. Rosenwaks Z, Liu HC, Picard JY et al. Anti-Miillerian hormone is not cytotoxic to human endometrial cancer in tissue culture. J Clin Endooinol Metab 1984; 59 : 166-169. 29. Wallen J, Cate RL, Kiefer DM et al. Minimal anti-proliferative effect of recombinant Mtillerian inhibiting substance on gynecological tumor cell lines and tumor explants. CcmcerRes 1989; 49 : 2005-2011. 30. George FW, Catt KJ, Neaves WB et al. Studies on the regulation of testosterone synthesis in the fetal rabbit testis. Endocff- 'nology 1978; 102 : 665-673. 31. Winter JSD, Faiman C, Reyes FI. Sex steroid production by the human fetus : Its role in morphogenesis and control by gonadotropins. In : Bergama DB., Blandau RJ, eds. Morphogenesis and Malformation of the Genital b'ystem. New York : Alan Liss, 1977, p 41. 32. Migeon BR, Brown TR, Axelman J ct al. Studies of the locus for androgen receptor localization on the human X chromosome and evidence for homology with the Tim locus in the mouse. Ptvc NatI Acad Sci USA 1981; 78 : 6339-6343. / 33. Lubahn DB, Joseph DR, Sullivan PM et al.
JOSSO : EMBRYOLOGY AND CLASSIFICATION OF INTERSEX STATE,S Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science 1988; 240 : 327-330. .34. Taguchi O, Cunha GR, Lawrence WD et al. Timing and irreversibility of M/illerian duct inhibition in the embryonic reproductive tract of the human male. Dev Biol 1984; 106 : 394-398. 35. Guerrier D, Tran D, VanderWinden JM et al. The persistent Mtillerian duct syndrome : a molecular approach. J Clin Endocnnol Metab 1989; 68 : 46-52. 36. Josso N, F6k6t6 C, Cachin O e t al. Persistence of M~illerian ducts in male pseudohermaphroditism, and its relationship to c~torchidism. Clin Endoctinol 1983; 19 : 247-258. 37. Siiteri PK, Wilson YD. Testosterone formation and metabolism during, male sexual differentiation in the human embryo. J Clin EndocdnolMetab 1974; 38:113-125. 38. Berihez6ne F, Forest MG, Grimaud JA et al. Leydig-cell agenesis : a cause of male pseudohermaphroditism. New Engl J Med 1976; 295 : 969-972. 39. Imperato-McGinley J, Guerrero L, Gautier T et al. Steroid 5 alpha-reductase deficiency in man : an inherited form of male pseudohermaphroditism. Science 1974; 186 : 1213-1215. 40. Keenan BS, Meyer WJ, Hadijian AJ et al. Syndrome of androgen insensitivity in man : absence of 5a-dihydrotestosterone binding protein in skin fibroblasts. J Clin Endoctinol Metab 1974; 38 : 1143-1146. 41., Brown TR, Lubahn DB, Wilson EM et al. Deletion of the steroid-binding domain of
42.
43.
44.
45.
46.
47.
48.
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the human androgen receptor gene in one family with complete androgen insensitivity syndrome : evidence for further genetic heterogeneity in this syndrome. Proc Natl Acad Sci USA 1988; 85 : 8151-8154. Forest MG, Cathiard AM. Pattern of plasma testosterone and delta 4-androstenedione in normal newborns : evidence for testicalar activity at birth. J Clin EndocHnolMetab 1975; 41 : 977-980. Corbier P, Kerdejhue B, Picon R et al. Changes in testicular weight, serum gonadotropin and testosterone levels before, during and after birth in the perinatal rat. Endocrinology 1978; 103 : 1985-1991. Maclusky NJ, Naftolin F. Sexual differentiation of the central nervous system. Science i981; 211 : 1294.1303. Forger NG, Breedlove SM. Sexual dimorphism in human and canine spinal cord : role of early androgen. Proc Natl Acad Sci USA 1986; 83 : 7527-7531. Money J, Hampson JG, Hampson JL. Hermaphroditism : recommendations concerning assignment of sex, change of sex, and psychologic management. Bull John Hopkins Hosp 1955; 97 : 284-300. Imperato-McGinley J, Peterson RE, Gautier T et al. Androgens and the evolution of male gender identity among male pseudohermaphrodites with 5a-reductase deftciency. New Engl J Med 1979; 300 ; 12331237. Jost A, Cressent M, Dupouy JP et al. Becoming a male. Adv Biosci 1972; 10 : 3-13.
I
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Embryology and Classification of lntersex States Nathalie Josso
Unite de Redterches stir L' Endocrinologie dtt Developpement, INSERa~I, I-t6pital des Enfants-Malades, Paris, France From the time of fertilization to birth, the mammalian conccptus develops from an egg to an individual characterized by a defined phcnotypic sex, either male or female. Sex differentiation can be defined as the "cascade" leading from genotypic to phenotypic sex. It is achieved before birth in all eutherian mammals. The pioneering experiments of Jost ~'2 have clearly illustrated the assymetrical basis of sex differentiation. Constitutively, the mammalian embryo is programmed to develop as a female, and male-determining agents are required to impose masculine characterislics on the body. The hinge between genetic and hormonal agents of male sex differentiation is located at the level of the fetal gonad: diverted towards testicular differentiation by Y-located genes, it produces in turn hormones which trigger male development of the internal and external reproductive tract. TESTICULAR DIFFERENTIATION In the mammalian fetus, the gonadal priReprint requests : Dr. Nathalie Josso, Unite de Recherches sur L' Endocrinologie du Dcveloppment, INSERM, Hopital des EnfantsMalades, Paris, France
mordium is represented by the gonadal ridge, a thickening of the coelomic epithelium covering tee anterior surface of the mesonephros, which is progressively colo, nized by primordial germ ceils travelling from the stalk of the allantois through the mesentery and the wall of the fetal gut ~. The first recognizable event of testicular differentiation is the development of a new cell type, the primordial Sertoli cells, which soon aggregate to form seminiferous tubules in which germ cells become enclosed46. In contrast to extratesticular germ cells, which, like female ones, enter the prophase of the first meiotic division early in fetal life7, spermatogonia enclosed in testicular tubules do not undergo meiosis until the beginning of puberty. In the human fetus, the testis can be recognized as early as 7 weeks after fertilization. Fetal Sertoli cells art large, clear cells, with abundant cytoplasm containing vesicles of rough endoplasmic reticulum. In these vesicles is: stored s, prior to secretion, a glycoprotein, anti-Miillerian hormone (AMIt), which is responsible for the inhibition of the development of the Miillerian ducts in male fetuses x,2, and which may also play a role in gonadal differentiation, as will be discussed later. Scrtoli cells continue to produce AMH throughout the whole of
This article is based on the presentation in the "International Workshop on Recent Advances in Neonatal Surgery and Intcrsex Disorders" hcld at All India Institute of Medical Sciences, New Delhi from March 1-4, 1989.It was accepted for publication in 1991.
468
THE INDIAN JOURNAL OF PEDIATRICS
gestation, and in the immediate postnatal period (reviewed in relY). Leydig cells differentiate somewhat later, at 8 weeks of gcstation in the human fetus. Their number increases dramatically until 14 to 16 weeks, and then Lcydig cells degenerate, and just prior to birth, very few are still visible in the interstitial tissue. Fetal Leydig cells, stimulated by chorionic gonadotropin, produce testosterone, which is responsible for the male dcvclopment of Wolffian derivatives, urogenital sinus and external genital organs. Agents of Teslieular Differentiation
Vol. 59, No. 4
hunt for Z F Y / Z F X related sequences had led to the disquieting mapping of homologous sequences of autosomal DNA of marsupials t4 implying that ZFY is not the testisdetermining gene in this therian subclass, which divcrged from placental mammals 45 million ycars ago. SRY does not act directly upon gonadal sex differentiation. The structure of the encoded protein is typical of one with DNAbinding affinity. Since testicular sex differentiation is a complex process, inw)lving also autosomal genes ~5, the protein encoded by SRY could play a regulatory role by activating or inhibiting the transcription of other genes. Proteins encoded by such "downstream genes" should exert a testis-determining activity per se. In this regard, it may be interesting to note that anti-Miillerian hormone (AMH) has recently been shown to mascullnize the structure and function of fetal ovaries in vitro 16,x7and is therefor.-, a candidate for SRY regulation. Fetal ovaries exposed to AMH in vitro divert their hormonal output from estrogen to testosterone production ~8.
What induces the fetal gonad to develop into a testis? The critical role of the Y chromosome in mammals has been recognized for a number cf years: regardless of the number of X chromosomes in their karyotype, cmbryos carrying a Y chromosome develop as males. This rule, howevcr, is not absolute : some XX individuals develop into males ~~ in contrast, chromosomal males with a female phenotype have been identified n. The genetic analysis of the DNA of these patients, pursued by a posse of investigators has becn crowned by the isolation FETAL TESTICULAR HORMONES of a sex determining gene, SRY ~2, coding for a DNA-binding protein, often called tesAs reviewed above, testis-determining tis-determining factor (TdF; referred to as genes are responsible for diverting an emTdy in the mouse). The SRY probe detects bryonic gonad from a constitutive female male-specific sequences in most-but not all- pathway towards testicular differentiation. XX males, but does not hybridize to Testis-determining genes are not involved DNA of XY females, leading to the conclu- in somatic sex differentiation, which is insion that translocation or deletion of this duced by fetal testicular hormones, regardDNA sequence is responsible for the appar- less of genotype. The classical experiments ent discrepancy between sex genotype and of Jost 2s have clearly demonstrated that the phenotype. SRY is highly conserved, and fetal ovaries play no active part in somatic shows similarities with products of yeast sex differentiation: agonadal fetuses differgenes involvcd in mating. A previous candi- entiate normally as females. Thus, the date for TDF, a zinc-finger transcription assymetrical nature of sex differentiation is unit (ZFY) had been put forward ~3but the reiterated: the testes must impose mascu-
JOSSO : E M B R Y O L O G Y A N D CLASSII:ICATION OF I N q ~ R S E X STATES
linity upon a body which would otherwise develop along female lines. Failure of the testes to carry out their mission-through incapacity to synthesize the appropriate hormones, or through end-organ resistance to these hormones-leads to male pseudohermaphroditism. As demonstrated by Jost 2, two discrete hormones are synthesized by the fetal testis : the Miillerian inhibitor (AMH) and testosterone. Anti-Miillerian Hormone
Anti-Miillerian hormone (AMH) is a 145,000 dimer containing 13.5% carbohydrate synthesized by immature Sertoli cells s and also by adult granulosa cells (see ref9 for review). Bovine testicular AMH has bccn purified to homogeneity by immunochromatography on a monoclonal antibody19, and the gencs coding for bovine and human AMH 2~have been cloned. The gene has been localized to the tip of the short arm of human chromosome 192I. The C-terminal part of the molecule shares a # 30% homology with the beta subunit of bovine inhlbin, with transforming growth factor beta 2~ whose genc has also been mapped to human chromosomQ 19, with the product of the decapentaplegic complex of Drosophila 22 and the Vgl gene of Xenopus 23. Proteolytic processing of recombinant AMH (MIS) produces a transforming growth factor-beta-like fragment ~. AMH is responsible for the regression of Miillerian derivatives in the male fetus 1'2 and has also been shown to masculinize fetal ovaries (see above). According to Hutson and Donahoe z~ AMH could also promote testicular descent, but no experimental evidence for this hypothesis has been provided. To the contrary, Fentener van Vlissingen et a126 have shown that AMH does not stimulate the growth of guber-
469
naculum cells in vitro, whereas a fetal testicular extract is active in this system. Partially purified bovine AMH has been reported to exert art antiproliferative effect on malignant cell lines of gynecological origin 27 but this has not been confirmed using an hormone purified to homogeneity~. Human recombinant AMH has no anti-cancer activity 29. Testosterone
Testosterone is produced by fetal Leydig cells from the time of their differentiation at 8 weeks of age. Initiation of testosterone secretion requires no hormonal sfimulation, but continued production requires the presence of gonadotropin 3~ which explains why the serum levels of testosterone and hCG are well correlated in the human fetus31. Testosterone exerts its biological effect through binding to an X-linked receptor molecule3z, which has a much greater affinity for dihydrotestosterone (DHT) than for testosterone itself. Therefore, in tissues containing alpha-reductase, the enzyme metabolizing testosterone to DHT, DHT is in fact the active androgen. The gene coding for the androgen receptor has recently been cloned from a flow-sorted human chromosome library, using as probe a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors 33. The deduced aminoacid sequence for the DNA-binding domain of the androgen receptor eDNA closely resembles that of the progesterone, mineralocorticoid and glucocorticoid receptor, and contains cysteine residues which could be involved in "finger" formation. NORMAL AND ABNORMAL MALE SOMATIC SEX DIFFERENTIATION Normal male somatic sex differentiation
470
THE INDIAN JOURNAL OF PEDIATRICS
is characterized by regression of Miallerian derivatives, maintenance and differentiation of the Wolffian ducts into vasa deferentia, epididymes and seminal vesicles, and the virilization of the urogenital sinus and external genitalia. Miillerian Duct Regression
AMH-mcdiated Miillerian duct regression begins at 8 weeks in the human fetus, and is complete at approximately 9 weeks34. Relatively high levels of AMH have been detected in the serum of fetal ruminants and human males up to puberty (reviewed in reference 9). In the human fetus, AMH could act by local diffusion from the testis to the adjacent Miillerian duct : in alternating true hermaphroditism, and in mixed gonadal dysgenesis, the unilateral testis has no inh!bitory effect upon the contralateral Miillefiah duct. Being a polypeptide hormone, AMH probably affects its target organ through binding to a membrane receptor molecule, but this hypothesis has not yet been borne out by experimcntat proof. Abnormal persistence of Mtillerian derivatives has been described in otherwise normal males35. The molecular basis of this condition is heterogeneous : in some, but not all, patients, immature testicular tissue expresses AMH normally, suggesting that the persistent Miillerian duct syndrome, a genetically transmitted defect, could be due to mutations affecting the biosynthesis of either the hormone itself, or of its putative receptor. In other instances, persistence of MiJllerian derivalives is associated with impairment of testosterone-dependent steps of sex differentiation, and is a consequence of testicular dysgenesis36.
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The Woiffian Ducts
Wolffian ducts, initially the excretory ducts of the primitive mesonephros, become testosterone dependent later, when they become part of the male genital system. In humans, Wolffian ducts depend for their differentiation essentially upon the amount of testosterone they can take up locally (33). Testosterone itself, and not DHT, is the active androgen, since fetal Wolffian ducts contain no 5-alpha-reductase37. Urogenital Sinus and External Genitalia
Virilization of these structures is initiated at 10 weeks in the human fetus. It is characterized by the inhibition of the development of the vaginal plate, the development of prostatic buds, the growth of the genital tubercle and the closure of the urogenital folds. In these tissues, testosterone is reduced to DHT by 5-alpha reductase, binds to intracellular androgen receptors which then activate the transcription of virilizing genes. Incomplete external virilization can be due to various causes. Defects in testosterone synthesis by the fetal testis may be caused by Leydig cell agencsis38, generalized testicular dysgenesis3~, or may arise from congenital defects of steroidogenesis. Peripheral insensitivity to testosterone may be caused by defects of 5-alpha-reductase~9, or by androgen receptor defects4~ Recently, deletion of the steroid-binding domain of the human androgen receptor gene has been reported in a family with a receptor(-) form of the complete androgen insensitivity syndrome41. The cause of androgen insensitivity in patients cxhibiting quantitatively normal binding of androgen in cultured fibroblasts remains to be determined.
JOSSO : EMBRYOLOGY AND CLASSIFICATIONOF INTERSEX STATES Sex Differentiation of the Brain
Differentiation of neuroendocrine patterns and sex behaviour is regulated by testosterone during the neonatal period. In both humans 4z and rats 43, a peak of testicular activity is observed immediately after birth. As reviewed by MacLusky and Naftolin 44, neonatal and androgenization of rodents induccs a permanent change in hypothalamic function which leads to the suppression of the LH midcycle surge. Testosterone treatment also abolishes the sexual dimorphism of the spinal cord 45. In contrast to neuroendocrine sex differentiation, psychological sex differentiation in humans is thought to be acquired postnatally by sociologic imprinting on the developing personality 46. However, the determining influence of the sex of rearing has been questioned, and lmperato-McGinley et a147 believe that an initially female gender identity can evolve at puberty under the influence of rising androgen production by the testes. In conclusion, because disorders of sex differentiation do not seriously impair the somatic heallh of their victims, clinical studies provide "experiments of nature" to illustrate physiological hypotheses. This field invites the continued collaboration of physicians and basic scientists to unravel the last mysteries obscuring what Jost 48 has called a prolonged, uneasy and risky venture : becoming a male! REFERENCES i. Jost A. Recherches sur la diffdrenciation sexuelle de l'embroyn de lapin, lII. R61e des gonades foetales dans la diff6renciation sexuelle somatique. Arck Anat Mierosc Motphol E W 1947; 36:271-315. 2. Jost A. Problems of fetal endocrinology :
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the gonadal and hypophyseal hormones. Recent Progrtlonn Res 1953; 8 : 379-418. 3. McKay DG, Hertig AT, Adams EC et al. HistochemicaI observations on the germ cells of human embryos. Anat Ree 1953; 117 : 201-219. 4. Agelopoulou R, Magre S, Patsavoudi E e t al. Initial phases of the rat testis differentiation in vitro. J Embryol Exp Motphol 1984; 83 : 15-31. 5. Jost A. Donn6es pr61iminaires sur les stades initiaux de la diff6renci.ation du testicule chez le rat. Atvh Anat Microse Morphol Exp 1972; 61 : 415-437. 6. Magre S, Jost A. The initial phases of testicular organogenesis in the rat. An electron microscopy study. Arch Anat Mictvsc Morphol Exp 1980; 69:297-318. 7. Upadhyay S, Zamboni L. Ectopic germ cells - Natural model for the study of germ cell sexual differentiation. Proc Natl Acad Sci USA 1982; 79 : 6584-6588. 8. Tran D, Josso N. Localization of anti-Mfillerian hormone in the rough endoplasmic reticulum of the developing bovine Sertoli cell using immunocytochemistry with a monoclonal antibody. Endoclinology 1982; 111 : 1562-1567. 9. Josso N, Care RL, Picard JY ct al. AntiMfillerian hormone, the Jost factor. Recent Progrllotm Res 1992; 48 (in press). 10. De la Chapelle A, Koo GC, Wachtel SS. Recessive sex-determining genes in human XX male syndrome. Cell 1978; 15 : 837-842. 11. Disteche CM, Casanova M, Saal H c t al. Small deletions of the short arm of the Y chromosome in 46, XY females. Proc Natl Acad Sci USA 1986; 83 : 7841-7844. 12. Sinclair AH, Berta P, Palmer MS et al. A gene from the human sex-determining region encodes a protein with homology ~o a conserved DNA-binding motif. Nature 1990; 346 : 240-244. 13. Page DC, Mosher R, Simpson EM et al. The sex-determining region of the human Y chromosome encodes a finger protein. Cell 1987; 51 : 1091-1104.
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