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Ovid: Clinical Gynecologic Endocrinology & Infertility

Editors: Speroff, Leon; Fritz, Marc A. Title: Clinical Gynecologic Endocrinology & Infertility, 7th Edition Copyright 2005 Lippincott Williams & Wilkins
> Table of Contents > Part II - Clinical Endocrinology > 9 - Normal and Abnormal Sexual Development > Normal Sexual Differentiation

Normal Sexual Differentiation


Part of "9 - Normal and Abnormal Sexual Development" The gender identity of a person (whether an individual identifies as a male or a female) is the end result of genetic, hormonal, and morphologic sex as influenced by the environment of the individual. It includes all behavior with any sexual connotation, such as body gestures and mannerisms, habits of speech, recreational preferences, and content of dreams. Sexual expression, both homosexual and heterosexual, can be regarded as the result of all influences on the individual, both prenatal and postnatal. Specifically, gender identity is the result of the following determinants: genetic sex, gonadal sex, the internal genitalia, the external genitalia, the secondary sexual characteristics that appear at puberty, and the role assigned by society in response to all of these developmental manifestations of sex.

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P.320 Prenatally, sexual differentiation follows a specific sequence of events. First is the establishment of the genetic
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sex. Second, under the control of the genetic sex the gonads differentiate, determining the hormonal environment of the embryo, the differentiation of internal duct systems, and the formation of the external genitalia. It has become apparent that the embryonic brain is also sexually differentiated, perhaps via a control mechanism very similar to that which determines the sexual development of the external genitalia. The inductive influences of hormones on the central nervous system can have an effect on the patterns of hormone secretion and sexual behavior in the adult.1, 2, 3, 4, 5, 6, 7, 8

Gonadal Differentiation
Both the X and the Y chromosomes evolved from autosomal ancestors in a 300-million-year time line.9 Most of the ancestral genes on the Y chromosome deteriorated, leaving only a limited number of currently active genes. The male-specific area of the Y chromosome encompasses almost all of the active genes (small parts are identical to corresponding regions of the X chromosome), and most importantly, the Y chromosome contains the gene that is essential for testicular development. In human embryos, the gonads begin development during the fifth week of gestation as protuberances overlying the mesonephric ducts. The migration of primordial germ cells into these gonadal ridges occurs between weeks 4 and 6 of gestation. Although germ cells do not induce gonadal development, if the germ cells fail to arrive, gonads do not develop and only the fibrous streak of gonadal agenesis will exist (Chapter 3). At 6 weeks of gestation (4 weeks after ovulation) the gonads are indifferent but bipotential, possessing both cortical and medullary areas and capable of differentiation into either testes or ovaries. They are composed of germ cells, special epithelia (potential granulosa/Sertoli cells), mesenchyme (potential theca/Leydig cells), and the mesonephric duct system. Wolffian and mllerian ducts exist side by side; external genitalia are undifferentiated.10 Subsequent sexual differentiation requires direction by various genes, with a single gene determinant on the Y chromosome (testes-determining factor, TDF) necessary for testicular differentiation, beginning at 67 weeks gestation.11

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P.321 About 95% of the length of the Y chromosome, previously known as the non-recombining region and now the male-specific region, encodes 27 distinct proteins that are male-specific, involved in testicular function.9 Deletions in this area are recognized as a cause of failure in spermatogenesis. Approximately 1015% of this area consists of material that originated in the X chromosome over the past few million years, and another 20% that came from the X chromosome even more distantly in the past. The distal ends of the short arms of the X and Y chromosomes are called the pseudoautosomal regions because during meiosis the homologous distal short arms of the X and Y chromosomes pair, and interchange of genetic material occurs as in autosomes. There are actually two pseudoautosomal regions on the Y chromosome, one at the terminal region of the short arm and one at the end of the long arm, but exchange of genetic material is more common with the short arm.12 The genes in the pseudoautosomal regions are doubly present in both sexes, and therefore escape X inactivation. Gene deletions in this area of the X chromosome (Xp22.3) are associated with various conditions, known as contiguous gene P.322 syndromes: short stature, mental retardation, X-linked ichthyosis, Kallmann's syndrome. The testes-determining gene is located on the distal short arm of the Y, immediately adjacent to the pseudoautosomal region. Loss of the TDF gene causes gonadal dysgenesis. Transfer of the TDF gene to the X results in an XX male. Since the identification of the Y chromosome's importance to male differentiation over 4 decades ago, 3 proteins have been suggested as the Y-encoded, gene-expressed, testes-determining factor. The first was the H-Y histocompatibility antigen and the second, ZFY (a zinc finger protein). Both were abandoned because of inconsistencies of expression in various cell types (in XX males and XY females), as well as absent expression in indisputable males with testes. SRY (Sex determining Region Y) is almost certainly the true sex-determining region on the short arm of the Y chromosome, the only gene on the Y chromosome required for sex determination.13 SRY is a single exon gene located in the smallest Y chromosome region capable of sex reversal. It is expressed in the genital ridge only during the appropriate time of embryonic development when testicular cords form; it is deleted or mutated in cases of human XY females; it is present in 46,XX males, and it can sex-reverse XX mice into males.14, 15, 16, 17 The 204 amino acid protein product contains a 79 amino acid domain with a motif shared by a recognized family of transcription factors (the high mobility group, HMG) that bind to DNA and regulate gene transcription. The high mobility group of transcription factors operate by binding and bending DNA, a dynamic, conformational mechanism. Investigations of the DNA-binding properties of the protein of SRY in the promoter regions of P450 aromatase (conversion of testosterone to estradiol that is down-regulated in the male embryo) and anti-mllerian hormone (responsible for regression of the mllerian ducts) support the conclusion that SRY directly controls male development through sequence-specific regulation of target genes.18 The HMG box provides the proteins encoded by SRY and its close relatives, the SOX genes. After binding to DNA, normal function is dependent on the subsequent alteration in DNA architecture. Mutations that abolish the ability of SRY protein to bind to DNA or to change the angle produced after DNA binding eliminate male differentiation.19 The expression of SRY in the tissue destined to become a gonad directs the cells of this gonadal primordium to differentiate as Sertoli cells. SRY participation in morphogenesis leading to a testis from the bipotential genital ridge is a model genetic switch between alternative inherent programs. Whereas testes formation is an active
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event, female sex determination has been considered to be the default pathway occurring if SRY is absent or deficient. The default concept is not entirely accurate. Mice with deletions of either the estrogen receptor genes or the aromatase genes indicate that estrogen activity is necessary to produce a normal ovary without male-like cells.20 In the total absence of estrogen receptors, mouse granulosa cells express SOX9 and differentiate into functional Sertoli cells.21 A recognized sequence of events is derived from studies of the mouse.19, 22, 23 SRY expression begins in the sexually indifferent gonadal ridges. The initial target cells are those destined to differentiate into either Sertoli cells or granulosa cells. The expression of SRY at this critical time moves differentiation along the male pathway to Sertoli cells, and it is believed that the Sertoli cells direct the other cell types into the appropriate testicular differentiation. At least two genes are important at this early stage, SOX9 and DAX1. The SOX (SRY-like box) genes are similar in sequence to SRY, but are not located on the Y chromosome; DAX1 is named for the location of its gene on the X chromosome, Xp21.3. In males, expression of SRY is immediately followed by activation of SOX9 that persists throughout testis development. In females, the absence of SRY sustains the inactivity of SOX9. DAX1 activity follows the opposite pattern: maintenance of high protein expression in the female gonad and switching off when SOX9 expression increases in the male gonad. However, in the mouse knockout model, some DAX1 expression is required for normal testis differentiation.24 The target cells for the protein expression of SRY, SOX9, and DAX1 are indifferent cells destined to proliferate and become Sertoli or granulosa cells. In the male, SOX9 expression is rapidly followed by activation of transcription of the anti-mllerian hormone (AMH).

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DAX1 is involved in both early sex determination and normal steroidogenesis. Mutations in the DAX1 gene in males are the cause of adrenal insufficiency in the first months of life, but also late-onset X-linked adrenal hypoplasia congenita, manifested clinically with hypogonadotropic hypogonadism, varying degrees of adrenal failure, and azoospermia.25 Female carriers of DAX1 mutations may experience delayed puberty.26 Genes other than SRY are also required for proper gonadogenesis.27, 28 In the human, autosomal genes are essential for gonadal development. These autosomal genes regulate migration of the germ cells and coding of the steroidogenic enzymes. The formation of the testicle precedes any other sexual development in time, and a functionally active testis controls subsequent sexual development; therefore, SRY presumably controls these autosomal genes. Thus, testicular hormones activate or repress genes to direct development away from an otherwise predetermined course of female differentiation. Steroidogenic factor-1 (SF-1) and DAX-1 are nuclear receptors for which specific ligands have not been identified (orphan receptors). SF-1 influences the expression of genes that encode steroidogenic enzymes and antimllerian hormone, and when genetic expression of SF-1 is disrupted in mice, gonads and adrenal glands fail to develop.29, 30, 31 SF-1 (in the mouse, the gene encoding SF-1 has been designated as FTZF1) expresses SF-1 in the genital ridges, therefore, to direct gonadal differentiation. Later, its expression in the Leydig cells is important for testosterone production and in the Sertoli cells for the production of anti-mllerian hormone.32 Mutations in the DAX1 gene result in adrenal hypoplasia, and DAX-1 is believed to work with SF-1 in regulating development and function of steroid-producing tissues.33 SF-1 and DAX-1 interactions are complicated and necessary for the transcription of multiple genes involved in normal sex determination and normal adrenal development and function. P.324 The WT1 gene is named after the Wilms' tumor nephroblastoma because it is one of the genes on chromosome 11 deleted in patients with this tumor. Mutant mice lacking WT1 fail to develop kidneys and gonads. WT1 mutations, however, could not be detected in 25 patients with a congenital absence of the uterus and vagina, indicating that WT1 is necessary for normal renal and gonadal development, but not for early mllerian duct development.34 Phenotypic girls with renal disease may have a WT1 deficiency and an XY genotype. Testicular differentiation begins at 67 weeks first with Sertoli cells that aggregate to form spermatogenic cords, then seminiferous tubules, followed by Leydig cell formation a week later. The Sertoli cells are the sites of SRY expression; therefore, the Sertoli cells orchestrate the development of primordial germ cells into testes. The molecular sequence of events may follow this scenario: SRY activation follows early expression of SF-1. SRY expression raises SF-1 and SOX9 activity to a critical level in the Sertoli cells, causing male differentiation and activation of the AMH gene. In females, maintenance of high levels of DAX1 expression antagonizes SF-1 action, causing SF-1 and SOX9 to switch off. This inhibition of the male pathway also involves another gene expressed specifically in XX gonads, Wnt4.23 Human chorionic gonadotropin (hCG) stimulation produces Leydig cell hypertrophy, and peak fetal testosterone levels are seen at 1518 weeks of pregnancy.27 It has been suggested that hCG stimulates steroidogenesis in the early fetal testes, so that androgen production will ensue and masculine differentiation can be accomplished.35 However, normal masculine differentiation occurs in mouse models lacking luteinizing hormone (LH) receptors, and molecular evidence indicates that fetal Leydig cells (but not adult cells) respond to adrenocorticotropic hormone (ACTH) as well as hCG.36 A primary role for ACTH is supported by the report of a male with an inactivating mutation of gene for the hCG/LH receptor who developed female genitalia (lack of AMH) along with a vas deferens and epididymis (testosterone stimulation).37 In an XX individual, without the active influence of a Y chromosome, the bipotential gonad develops into an ovary about 2 weeks later than testicular development. The cortical zone develops and contains the germ cells, whereas the medullary portion regresses with its remnant being the rete ovarii, a compressed nest of tubules and Leydig cells in the hilus of the ovary. Normal ovarian differentiation requires the presence of the germ cells, indicating some form of communication between the germ cells and the somatic cells. The germ cells proliferate by mitosis, reaching a peak of 57 million by 20 weeks of pregnancy. By 20 weeks, the fetal ovary achieves
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mature compartmentalization with primordial follicles containing oocytes, initial evidence of follicle maturation and atresia, and an incipient stroma. Degeneration (atresia) begins even earlier, and by birth, only 12 million germ cells remain. These have become surrounded by a layer of follicular cells, forming primordial follicles with oocytes that have entered the first meiotic division. Meiosis is arrested in the prophase of the first meiotic division until reactivation of follicular growth that may not occur until years later. Excessively rapid atresia (germ cell attrition) in gonadal dysgenesis (45,X) accounts for the streak gonad seen in these cases.38 A complete 46, XX chromosomal complement is necessary for normal ovarian development.39 The second X chromosome, therefore, contains elements essential for ovarian development and maintenance. Summary of key genetic events in early sex determination:
q

Migration of primordial germ cells to the urogenital ridge.


q

Differentiation of the bipotential gonadal tissue under the direction of WT1 and SF-1.
q

SRY activation of male-specific genes, especially SOX9, to produce the testes by cell proliferation, differentiation, migration, and vascularization.
q

Ovarian differentiation by suppression of SOX9 through the activity of DAX1 and Wnt4.

P.325 The genetics of gonadal dysgenesis can be summarized as follows:


q

Gonadal streaks without germ cells in XX or XY individuals (female phenotype):


r

Deficiencies in WT1 or SF-1


q

Lack of testicular development in XY individuals, pure gonadal dysgenesis (female phenotype):


r

Deficiencies in SRY or SOX9


q

Male phenotype in a 46,XX individual:


r

Presence of SRY
q

Mixed gonadal dysgenesis in mosaics (varying phenotype):


r

Excess DAX1

Duct System Differentiation


Caspar Wolff described the mesonephros in 1759 in his doctoral dissertation when he was 26 years old.40 The paired structures of the mesonephros of the early vertebrate embryo were named wolffian bodies by the nineteenth century embryologist, Rathke, in recognition of Wolff's initial discovery and description. Johannes
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Mller, a German physiologist with a prodigious academic output, described the embyrology of the genitalia in 1830. The paramesonephric ducts received his name, not because of his original contributions, but because of his ability to synthesize current knowledge in his effective writings. His physiology text was a standard in many European countries. Renal development goes through 3 stages: pronephric, mesonephric, and metanephric. The mesonephric ducts remain for development as internal genitalia. At this stage the mesonephric ducts are called the wolffian ducts. The paired paramesonephric ducts are the mllerian ducts. The wolffian and mllerian ducts are discrete primordia that temporarily coexist in all embryos during the ambisexual period of development (up to 8 weeks). Thereafter, one type of duct system persists normally and gives rise to special ducts and glands, whereas the other disappears during the third fetal month, except for nonfunctional vestiges. Hormonal control of mammalian somatic sex differentiation was established by the classic experiments of Alfred Jost.41 In Jost's landmark studies, the active role of male determining factors was defined as the directing feature of sex differentiation. This principle applies not only to the internal ducts but to the gonad, external genitalia, and even the brain. The critical factors in determining which of the duct structures stabilize or regress are the secretions from the testes: testosterone and anti-mllerian hormone (AMH), also known as mllerianinhibiting substance or mllerian-inhibiting factor. AMH is a member of the transforming growth factor- family of glycoprotein differentiation factors that includes inhibin and activin.42, 43 The gene for AMH has been mapped to the short arm of chromosome 19, 19p13.3. AMH operates through two receptors, first binding to the AMH type II receptor (expressed by its gene on chromosome 12q13), and then recruiting the signal transducer, the AMH type I receptor. This signaling system uses binding to a bone morphogenetic protein (BMP) receptor followed by activation of Smad proteins.44 AMH is synthesized by Sertoli cells (activated by SRY and SOX9) soon after testicular differentiation and is responsible for the ipsilateral regression of the mllerian ducts that proceeds in a craniocaudal direction and is complete by 8 weeks, before the emergence of testosterone and stimulation of the wolffian ducts.45 Despite its presence in male serum up to puberty, lack of regression of the uterus and tubes is the only consistent expression of AMH gene mutations. Knockout mice for AMH develop normal testes and ovaries.46 In the absence of AMH, the fetus will develop fallopian tubes, uterus, and upper vagina from the paramesonephric ducts (the mllerian ducts). This development requires the prior appearance of the mesonephric ducts, and for this reason, abnormalities in the renal system are associated with abnormalities in development of the tubes, uterus, and upper vagina. P.326 AMH has extra mllerian functions. AMH exerts an inhibitory effect on oocyte meiosis, plays a role in the descent of the testes, and inhibits surfactant accumulation in the lungs.47 Proteolytic cleavage of AMH produces fragments that have the ability to inhibit growth of various tumors (a potential therapeutic application). Testicular descent occurs in stages. Transabdominal movement of the testes is the result of rapid gubernacular growth, apparently under AMH control. Movement through the inguinal canal is mediated by androgens. In females, AMH is not expressed prior to birth, guaranteeing normal female differentiation. After puberty, AMH, produced by small growing ovarian follicles, is a paracrine inhibitory factor, perhaps preventing the excessive recruitment of resting primordial follicles, ensuring the emergence of only one dominant follicle.48 AMH, secreted by the Sertoli cells, is detectable in the serum of males during infancy, childhood, adolescence, and adulthood (with a decline to barely detectable levels after puberty). In contrast, AMH, secreted by granulosa cells, is not measurable until puberty in females.49 This difference allows serum measurement to be a sensitive marker for the presence of testicular tissue in intersex anomalies.50, 51 After puberty, testosterone, with an added contribution from meiotic germ cells, suppresses AMH secretion in males; therefore, individuals with the androgen insensitivity syndrome (androgen receptor defects) have very high AMH levels after puberty.52 The failure of testosterone to suppress AMH secretion during fetal and newborn life is explained by the absence of the androgen receptor in Sertoli cells until later in life. Testosterone is secreted by the fetal testes soon after Leydig cell formation (at 8 weeks) and rapidly rises to

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peak concentrations at 1518 weeks of pregnancy. This testosterone secretion stimulates development of the wolffian duct system into epididymis, vas deferens, and seminal vesicles. Testosterone levels in the male fetus correlate with Leydig cell development, overall gonadal weight, 3-hydroxysteroid dehydrogenase activity, and chorionic gonadotropin (hCG) concentrations. As hCG declines (approximately 20 weeks) the fetal pituitary luteinizing hormone (LH) assumes control of Leydig cell testosterone secretion; anencephalics and other forms of congenital hypopituitarism display diminished androgen effects on internal and external genitalia. The wolffian ducts receive testosterone signals directly from nearby Leydig cells as well as the general fetal circulation. This local paracrine effect is essential to the stimulation of ipsilateral differentiation into the epididymis, vas deferens, and seminal vesicles. Duct system differentiation will proceed, therefore, according to the nature of the adjacent gonad. The wolffian ducts do not form dihydrotestosterone, so the direct high concentration of testosterone is crucial for normal development.53 Because of this local paracrine action, wolffian development cannot be stimulated in females exposed to adrenal or exogenous androgens.

Figure. No caption available.

P.327 The internal genitalia possess the intrinsic tendency to feminize. In the absence of a Y chromosome and a functional testis, the lack of AMH allows retention of the mllerian system and development of fallopian tubes, uterus, and upper vagina. In the absence of testosterone, the wolffian system regresses. In the presence of a normal ovary or the absence of any gonad, mllerian duct development takes place. These classic roles for the presence and absence of testosterone are undisputed; however, estrogen may make a contribution in this process. Knockout mice for the estrogen receptor- have a marked retention of wolffian tubules, indicating that estrogen plays a role in the degeneration of the wolffian system.54

External Genitalia Differentiation


In the bipotential state (sixth gestational week), the external genitalia consist of a genital tubercle, a urogenital sinus, and two lateral labioscrotal swellings. Unlike the internal genitalia where both duct systems initially coexist, the external genitalia are neutral primordia able to develop into either male or female structures depending on gonadal steroid hormone signals. Normally, this differentiation is under P.328 the active influence of androgen from the Leydig cells of the testis. The genital tubercle forms the penis,
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labioscrotal folds fuse to form a scrotum, and folds of the urogenital sinus form the penile urethra. The testis begins androgen secretion by 89 weeks; masculinization of the external genitalia is manifest 1 week later and is completed by 14 weeks. To achieve this morphologic change, external genitalia target tissue cells must convert testosterone to dihydrotestosterone (DHT) by the intracellular enzyme 5-reductase. In the male, DHT mediates the following androgen events: temporal hairline recession, growth of facial and body hair, development of acne, and development of the external genitalia and prostate.

Figure. No caption available.

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In the absence of this androgen effect (the absence of a Y chromosome, the presence of an ovary, the absence of a gonad, abnormalities in androgen receptor or postreceptor events, or defects of the 5-reductase enzyme), the folds of the urogenital sinus remain open, forming the labia minora, the labioscrotal folds form the labia majora, the genital tubercle forms the clitoris, and the urogenital sinus differentiates into the vagina and the urethra. Thus, the lower vagina is formed as part of the external genitalia. In females, exposure to androgens at critical time periods leads to variable masculinization. Androgen exposure at 914 weeks superimposes variable external ambiguity on the basic female phenotype (clitoral hypertrophy, hypospadias, scrotalization of nonfused labia). By the same token, incompletely masculinized genitalia will result if sufficient local androgen concentration or activity is not achieved by the twelfth week in the male and maintained in the first postnatal months.55 Because of shared common tissue origin, male-female external genital structural ambiguities reflect abnormal androgen impact: males too little, females too much.

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Central Nervous System Differentiation (Gender Identity)


At the same time the presence or absence of androgens is playing a critical role in genitalia development, the neuroendocrine mechanism of the central nervous system (CNS) is also being influenced. Androgens present in sufficient amounts during the appropriate critical stage of development program the central nervous system to induce patterns of male sexual behavior.3, 5, 6, 7, 8, 56, 57, 58, 59 Experimental and analytical evidence indicates that a behavioral effect can be traced to this early androgen influence. Fetal hormonal programming contributes, therefore, to the spectrum of psychosexual behavior seen in humans. For this reason, the concept
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of gender neutrality at birth has been challenged and the wisdom of feminizing surgery for ambiguous genitalia questioned.60 Gender role is also heavily influenced by assignment of sex of rearing followed by social interaction based on genital appearance and the development of secondary sexual characteristics.

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