HUMAN SEXUAL DIFFERENTIATION
Division of Biology of Growth and Reproduction, Department of Pediatrics,
University Cantonal Hospital, 1211 Geneva 14, Switzerland
Fetal sexual differentiation is a very complicated series of events actively programmed, at appropriate critical periods of fetal life, which involves both genetic and hormonal factors leading to the sexual dimorphism observed at birth (Table 1). Sexual differentiation is achieved at midgestation. Genetic factors and hormonal factors will alternate in this chain of programmed transformations of the primary gonads, the internal sex structures and the external genitalia. Sex chromosomes promote the development and the differentiation of the primary gonad but the decisive influences are the presence or absence of testosterone and of antimüllerian hormone production by the testis. Femaleness results from the absence of any masculinizing genetic factor or hormone acting during the critical period of differentiation. Brain and hypothalamic sexual identities are mainly acquired during postnatal life. Gender and behaviour identities are markedly influenced by psychosocial imprinting.
Sexual differentiation is conformed in the human during four successive steps: the constitution of the genetic sex, the differentiation of the gonads, the differentiation of the internal and the external genital tractus and the differentiation of the brain and the hypothalamus.
The critical role of the Y chromosome and of male hormones in male orientation is well documented, the development of the female sexual differentiation occurring in the absence of male genetic determinants.
Genetic sex is established at the time of fecundation by the nature of the chromosomal composition of the spermatozoon, whether it contains a Y chromosome which has a dominant effect (23,Y constitution), or an X chromosome (23,X constitution). The development of such gonad into a testis depends upon the presence of the Y chromosome (59), whereas the absence of the Y chromosome will result in female development, irrespective of the number of X chromosomes (12,31,42). This effect was thought to be due to the presence of a unique gene located on the short arm of the Y chromosome (11).
Several years ago, such effect was supposed to be linked to a male-specific histocompatibility gene named the H-Y antigen (34), which was thought to be the primary testis-inducer (58). However it was soon found that such antigen did not explain all the sex-reversal cases observed in nature both in human and in mouse (47). Since then several genes have been proposed as candidates for the testis-determining factor (Fig. 1). An interval of 140 kb located between 140 and 280 kb of the proximal border of the pseudoautosomal pairing region was first isolated as the region with the testis-determining factor (TDF in the human and Tdf in the mouse) (35). Successive studies using recombinant DNA methods have tried to localize the TDF locus (Fig. 1). From a 140 KB region, a highly conserved gene was located in the 1A2 region of the Y chromosome, and named zinc finger protein-Y (ZFY), coding for zinc-finger-containing protein that could well function as a DNA-binding transcriptor regulator and be a good candidate for the testis-determining gene (28,36). However, an homologous sequence called zinc finger X (ZFX) was found on the X chromosome which questioned this hypothesis. From further deletion studies, it was found that the 1A1 region was the one most likely to contain the TDF gene. From this 60 kb region, a 35kb region was deducted, in which a single copy gene was found (48), that is highly conserved and shows homologies both with the sexual mating-type protein Mc required for mating in Schizosaccharides pombo yeast and with the nonhistone nuclear HMC (high mobility group) proteins expressed during embryogenesis (17,48); it was also thought to function as a DNA-binding transcription factor. This 14-kb gene has been named sex-determining region of the Y (SRY) in the human. The possibility remains that the ZFY and the SRY genes are two separate but neighbouring genes. However, testis development must only be possible through the interaction of Sry gene with other genes, located on autosomal chromosomes, some of which being involved in the regulation of Sry expression, others possibly being downstream targets of Sry (27).
The undifferentiated gonadal primordium, which is located at the ventral surface of the primitive kidney or mesonephros, is already visible in the 5 mm human embryo and consists of a thickening of the coelomic epithelium. In a first step, which is independent from the genetic sex, the gonadal primordium is colonized by the primordial germ cells originating from the allantoid sac. When these cells have reached the gonadal primordium, they form with the epithelium the " gonadal ridge ". The epithelium consists of two to three cylindric cells in which the gonocytes are present. This epithelium is separated from the mesonephros by a layer of mesenchymal cells. According to the classical Witschi’s theory, seminiferous tubules originate from the mesonephros, the " medulla ", while ovarian tissue originates from the secondary sex cords formed from the germinal epithelium or " cortex " (62). This theory is not universally accepted as recent observations have shown that the differentiation of the gonad occurs at same time of fetal age (7th week), for both the testis and the ovary (14).
The differentiation of the gonadal ridge into a testis is a rapid phenomenon, which contrasts with the slow and late development of the ovary. Testicular tissues, and in particular seminiferous tubules, are recognized in the human embryo at 7 weeks of fetal age (crown-rump length 13-20 mm) (21). Inside the seminiferous tubules, germ cells are large. They divide actively but do not enter meiosis. Sertoli cells are smaller than the germ cells. They tend to surround the germ cells and prepare the future seminiferous tubules. A basal membrane is formed which isolates the tubules from the surrounding mesenchymal tissue. The individualization of tubules and the synthesis of the antimüllerian hormone precede Leydig cells differentiation (53). Leydig cells differentiate from interstitial tissue, between the 8th and the 9th week (crown-rump length 32-35 mm), and spread progressively in the intertubular spaces between the 14th and the 18th week. They secrete testosterone from the 8th week (61). Maximal fetal serum concentration is observed from the 14th to the 16th week. Levels are comparable to those observed in adult males. After 20 weeks of gestation, Leydig cells involute, and circulating testosterone levels decrease progressively to levels observed in female fetuses. At birth, cord blood testosterone levels are higher in male newborns than in females (13).
Fetal testes localized in the kidney region start to descent at the 12th week of fetal age, reaching the internal orifices of the inguinal canal at midgestation, and finally the scrotum during the last two months of gestation. The mechanical and humoral factors involved in this process are still unclear. It is presently accepted that the transabdominal descent is not androgen-dependent. It may be due to the intraabdominal pressure and/or the traction exerted by the gubernaculum testis, whose development would be linked to the presence of a low molecular weight factor, named " descendin ", not yet well identified, but distinct from polypeptide growth factors and fetal testicular hormones (10). The transinguinal part of the descent is thought to be mainly androgen-dependent (20).
Orientation of the primordial gonad towards ovarian differentiation in XX subjects appears after the 2nd month of fetal age. Intense proliferation of the germ cells under the coelomic epithelium forms clusters which move inside the gonad constituting the Pflüger’s cords or ovarian cortex. From the 9th week, germ cells enter into the meiotic prophase. At the 16th week, the first ovarian somatic cells appear between the ovarian cortex and the central zone. They form granulosa cells which encircle the oocytes, blocked at the diplotene stage of the first meiosis. They will remain at this stage till ovulation. These structures are the first ovarian follicles. They can further develop with antrum formation and luteinization (43).
The role of the sex chromosomes in the differentiation of the ovary remains hypothetical. Inactivation of one of the X chromosome in the somatic 46,XX cells occurs at a very early stage of embryogenesis. In the oocytes, both sex chromosomes remain functional (15). Whether the two X chromosomes are necessary for the ovarian differentiation is still debated. Normal meiosis in 45,X female fetuses has been described and the disappearance of the germ cells from these ovaries is a late phenomenon, occurring after the 12th week of fetal age (18,21,49). Death of the germ cell induces degeneration of the follicle and loss of the endocrine activity of the ovary which is replaced by fibrous tissue or streak formation.
In addition, the oocytes remain able to migrate from the cortical layers of the ovary to the surface epithelium of the ovary and to be extruded and liberated into the peritoneal cavity throughout all stages of fetal ovarian development. At the 5th month of fetal age human fetal ovary contains 7 millions germ cells. At 7 months the human fetal ovary does not form any additional germ cells. At birth, this number has fallen to 2 millions and, at 7 years of age, to 300,000 (1). The different development of oogonia, which are blocked at the diplotene stage, and spermatogonia, which enter into meiosis at puberty, has not been fully explained. The germ cells are stimulated by meiosis-inducing factors secreted by both the male and fetal gonad (4). This stimulating action is counteracted by inhibiting factors secreted by Sertoli cells or granulosa cells (26,33). Spermatogonia are very early caught in a tight network of Sertoli cells which blocks meiosis. Oogonia develop as long as they are not surrounded by the granulosa cells.
The fetal ovary is capable of synthesizing estradiol as early as the 8th week of fetal age (16). Whether this secretion of estradiol plays a physiologic role in the human sex differentiation is not known. However, low expressions of mRNA for both P-450scc and P-450c17 enzyme activities have been observed in fetal ovaries (57), in contradiction with the possible local secretion of estradiol.
Hormones secreted by the fetal differentiated gonads induce the development of the internal and external genitalia.
Fetal Leydig cells produce testosterone in high amounts. There is circumstantial evidence that placental production of chorionic gonadotropin (hCG), which peaks at 12 weeks of fetal age, controls early fetal gonadal steroidogenesis (6). Testicular capacity of hCG binding is maximal at 15-20 weeks (32). Studies of the steroidogenic enzymes and of the expression of the steroidogenic enzyme genes have shown that cholesterol side-chain cleavage enzyme (P-450scc), adrenodoxin (iron-sulfur protein serving as an electron transport intermediate for P-450scc) and P-450c17 (17a-hydroxylase/17,20-lyase) genes are highly expressed in the fetal testis, mainly during the 14th and the 16th weeks. After 16 weeks, P-450c17 mRNA decreases more rapidly than P-450scc mRNA does (57). This age related pattern of P-450scc and P-450c17 mRNA is similar to fetal testicular and serum testosterone concentrations (44,45,52), and relates to the variations in hCG production and hCG receptors. These findings suggest that the expression of the steroidogenic genes is directly regulated by circulating hCG. The P-450Arom (aromatase gene) is poorly expressed in the fetal testis. The lack of significant local estradiol production may be an explanation for the non-desensitization of the fetal Leydig cells in the presence of high levels of hCG (29).
Fetal Sertoli cells produce the antimüllerian hormone (AMH, also named müllerian-inhibiting substance, or müllerian-inhibiting hormone). The existence of the antimüllerian hormone was postulated by Jost in 1947 (25). It is a glycoprotein of 145 kD molecular weight (40) which is secreted by the immature Sertoli cells, from early differentiation till puberty (7,22). Testicular production of AMH is maximal during the period of müllerian duct regression in males and decreases to a plateau throughout gestation (3,22). The role of AMH in later male development is not known, but an inhibitory effect on male germ-cell meiosis in fetal life, and a positive effect on testicular descent have been suggested (23). Human gene for AMH, which is located on chromosome 19, has been cloned and sequenced (5,41). AMH mRNA is readily detectable in human fetal testis with no significant change from 13 to 25 weeks of gestation (57). Proteolytic cleavage of AMH induces TGF-ß-like fragment. AMH has extramüllerian effects, such as an inhibiting action on the development of ovarian and uterine tumoral cells (8,23) and a virilizing action on fetal rat ovary, inducing structures similar to seminiferous tubules and secretion of testosterone instead of estradiol (55). The latter shift in steroidogenesis results from a repressor action of AMH on the biosynthesis of aromatase (56). This rises the possible role of AMH on the differentiation of the fetal testis as initiated by the SRY gene. In addition, AMH inhibits maturation of fetal lung cells, by its anti-EGF action. Persistent müllerian duct syndrome can be due to absence of AMH or to absent AMH receptors on target tissues.
Insulin-like growth factor I (IGF-I) concentration is weak in fetal testis (57). Conversely, insulin-like growth factor II (IGF-II) is abundant. IGF-II mRNA is present in large quantities. The developmental pattern of IGF-II mRNA expression is similar to that of the steroidogenic enzymes P-450scc and P-450c17: highest expression at 14-16 weeks and decrease thereafter (57). This pattern of IGF-II gene expression is not regulated in a similar way as that of the steroidogenic enzyme genes and could be only age-dependent.
Fetal ovary is able to convert androgens to estrogens in vitro (16). The physiological significance of the presence of aromatase in the fetal ovary remains unexplained, as the fetal ovary is lacking the other steroidogenic enzymes necessary for the synthesis of the steroid precursors (39). Aromatase mRNA is found at a weak level in the fetal ovary, fitting, however, with the observed fetal ovarian aromatase activity. In addition, during early gestation the fetal ovary does not contain hCG receptors (32) and its further development during late gestation may be dependent on the presence of pituitary gonadotropins (45). Gene expression of P-450scc, P-450c17 enzymes is very low in the fetal ovary (57). Fetal ovarian adrenodoxin mRNA abundance is about 50% of that of the testis (57). The significance of this relatively high adrenodoxin gene expression in a steroidogenically inactive fetal gonad remains unknown. AMH is not detectable in the fetal ovary. Only very small amounts of AMH mRNA can be detected in fetal ovarian tissue contrary to the adult ovary granulosa cells where AMH mRNA can be clearly detected (57). IGF-II mRNA can be also detected in the fetal ovary as well as in the fetal testis.
Differentiation of the internal and the external genitalia
The internal genitalia derives from the differentiation of two pairs of ducts: the wolffian ducts and the müllerian ducts (Fig. 2). Both ducts develop from the part of the mesonephros which does not participate to the formation of the fetal gonad. They both end in the urogenital sinus which opens to the perineum at the level of the urogenital orifice, located at the base of the genital tubercle.
Wolffian ducts are present in the embryo at a crown-rump length of 4-5 mm, and serve as the excreting duct to the mesonephros. When the definitive kidney becomes functional, the wolffian duct that is dependent of the presence of androgens becomes the vas deferens system. In the female fetus, the wolffian ducts degenerate. In the male fetus, the anterior part of the wolffian ducts communicate with the seminiferous tubules, the posterior part forms the vas deferens and the seminal vesicle. This differentiation is dependent of high local concentration of testosterone, which is only active during a " critical " period during which the wolffian duct is sensitive. Testosterone and not dihydrotestosterone, is the active hormone as the wolffian duct does not contain 5a-reductase activity at this stage of development (46). Testosterone receptors are present and their number increase with age. Development of the wolffian ducts can be partially inhibited by the injection of testosterone antibodies, or administration of cyproterone acetate to the pregnant animal (2,9). In male pseudohermaphroditism, the maintenance and differentiation of the wolffian ducts can be observed, because some testosterone is secreted very early and at low concentration, suggesting that their complete development is dependent of very high local concentration of testosterone.
Müllerian ducts appear in the human embryo at crown-rump length 10 mm. When the embryo is 50 mm, the uterovaginal canal formed by the reunion of both caudal terminals joins the posterior wall of the urogenital sinus, between the two orifices of the wolffian ducts. In the female embryo, müllerian ducts differentiate into fallopian tubes, the uterus and the upper part of the vagina. The uterine cervix develops later at crown-rump length 150mm. Receptors for estradiol have been found in the müllerian ducts (38,51), but their physiological significance is unknown as estrogens are not necessary for the differentiation of the female internal genitalia. In the male fetus, müllerian ducts begin to regress at crown-rump length 30 mm and have disappeared at crown-rump length 43 mm. This regression is due to the presence of the antimüllerian hormone (AMH). The müllerian duct is sensitive to AMH during a limited period of fetal development (up to 8 weeks in the human fetus). This hormone only acts locally.
Gonadectomy performed in male rabbit fetuses before the age of differentiation induces the degeneration of the wolffian ducts and the development of the müllerian ducts into fallopian tubes, uterus and the upper part of the vagina (24). In opposite experiments with female fetuses, locally implanted fetal testis induce the regression of the müllerian ducts and the development of the wolffian ducts. Local implants of testosterone induce development of the wolffian ducts and no regression of the müllerian ducts. These experiments led Jost to develop the concept of the two hormones: AMH and testosterone, influencing the differentiation of the male fetus (25).
Differentiation of the urogenital sinus and the external genitalia
In both sexes the urogenital and the external genitalia are similar up to the 9th week (crown-rump length 30 mm). The müllerian tubercle protrudes from the posterior wall of the urogenital sinus, between the two orifices of the wolffian ducts (Fig. 3). The external genitalia differentiate from the genital tubercle and the two lateral urethral folds and labioscrotal swellings (Fig. 4).
In the female fetus, vaginal organogenesis consists of the development of a vaginal plaque from the müllerian tubercle separating the müllerian vagina and the urogenital sinus. Later this vaginal plaque forms a canal (at crown-rump length 200 mm) and its upper part opens to the uterine cervix, while the müllerian vaginal epithelium regresses. Administration of high doses of estrogens to pregnant women prevents the progression of the vaginal epithelium from sinus origin. The persisting müllerian epithelium would be responsible for the vaginal adenosis frequently observed in girls who were submitted to high doses of estrogens in utero (54). The genital tubercle becomes the clitoris, the labioscrotal swellings do not fuse and the perineal anogenital distance does not increase.
In the male fetus, masculinization of the external genitalia begins at crown-rump length 43-45 mm. The vaginal plaque, which is small, forms the prostatic utricule. The urogenital sinus increases in length and forms the prostatic and the perineal urethra. The genital labioscrotal swellings fuse and the anogenital distance increases. At crown-rump length 90 mm (12th-14 weeks) the penile urethra is formed. The growth of the genital tubercle continues during gestation. The differentiation of the urogenital sinus and the external genitalia depends on the presence of the fetal testicle. In its absence, whether ovaries are present or not, the vagina develops and the labioscrotal swellings do not fuse. Testosterone is the hormone responsible for the male differentiation of the urogenital sinus and the external genitalia. However the presence of the 5a-reductase is necessary as the active metabolite on the external genitalia is dihydrotestosterone (46,60). The enzyme has been detected in these organs prior to their masculinization. Testosterone acts directly on the differentiation of the epididymis, the vas deferens and the seminal vesicle. Reduction of testosterone to dihydrotestosterone by 5a-reductase is necessary to obtain differentiation of the prostate, the prostatic utricule, the scrotum and the penis (Fig. 5). High dose of estrogens administrated in the pregnant animal may cause abnormal development of the male genitalia, leading to an intersex condition. Such anomalies have been described in human male neonates whose mothers have received diethylstilbestrol during pregnancy (19).
The concept proposed by Jost of an asymmetrical sex differentiation remains nowadays valid. It consists of a developmental pattern in which the " passive " female differentiation is counteracted by male genetic and hormonal factors, the sex-determining gene(s), and the two male hormones, testosterone and antimüllerian hormone. These factors and hormones act on target cells and tissues only during a " critical " period of development. The mechanism of this chronological " critical " period still lacks to date an adequate biological explanation.
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