Transcriptional Regulation of Gonadal Development and Differentiation
http://www.100md.com
内分泌学杂志 2005年第3期
Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: J. Larry Jameson, Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611. E-mail: ljameson@northwestern.edu.
Abstract
The embryonic gonad is undifferentiated in males and females until a critical stage when the sex chromosomes dictate its development as a testis or ovary. This binary developmental process provides a unique opportunity to delineate the molecular pathways that lead to distinctly different tissues. The testis comprises three main cell types: Sertoli cells, Leydig cells, and germ cells. The Sertoli cells and germ cells reside in seminiferous tubules where spermatogenesis occurs. The Leydig cells populate the interstitial compartment and produce testosterone. The ovary also comprises three main cell types: granulosa cells, theca cells, and oocytes. The oocytes are surrounded by granulosa and theca cells in follicles that grow and differentiate during characteristic reproductive cycles. In this review, we summarize the molecular pathways that regulate the distinct differentiation of these cell types in the developing testis and ovary. In particular, we focus on the transcription factors that initiate these cascades. Although most of the early insights into the sex determination pathway were based on human mutations, targeted mutagenesis in mouse models has revealed key roles for genes not anticipated to regulate gonadal development. Defining these molecular pathways provides the foundation for understanding this critical developmental event and provides new insight into the causes of gonadal dysgenesis.
LIKE MOST ORGAN systems, mammalian gonadal development involves a complex interplay of multiple cell types, and it occurs during a relatively narrow time window. Thus, it is important to understand the temporal pattern of gene expression as well as the spatial relationships of the developing tissues. For these reasons, most recent studies have used the mouse as a model for examining gonadal development because it is amenable to genetic manipulation as well as detailed histologic and biochemical characterization. During the transition from an undifferentiated gonad to a testis or ovary, the female and male gonad each display characteristic morphological features and patterns of gene expression (1). The coordinated differentiation of the embryonic urogenital ridge, from the bipotential state to sexual dimorphism, provides an opportunity to identify the key factors and commitment steps that underlie gonadal differentiation (2).
In humans, the functional significance of some of these molecules is evident from mutations that impair gonadal development and reproduction (3, 4, 5). For example, deletions of the Y chromosomal gene, SRY (sex-determining region on the Y chromosome) cause XY male-to-female sex reversal, whereas SRY translocations to the X chromosome lead to XX female-to-male sex reversal (6). The role of Sry as a candidate testis-determining gene was confirmed by demonstrating testis development after transgenic expression of Sry in XX mice (7). Thus, by using animal models, it is possible to explore genetic pathways in greater detail and elucidate disease pathogenesis.
Many of the gonadal development factors described to date are known to act at the transcriptional level. For the most part, their functions are incompletely understood. Based on homology to other transcription factors, some affect DNA bending (8) or modulate chromatin remodeling (9). Others form interactive complexes that activate transcription (10) or have a role in specifying progenitor cell types (11). Inhibition of gene expression is equally likely to be important as a means to dictate cell fate, but less is known about potential transcriptional repressors. The functional interaction between extracellular ligand molecules and nuclear transcription factors also merits emphasis. For example, extracellular signals can induce transcription factor release and translocation to the nucleus [i.e. ?-catenin/lymphoid-enhancing factor, Smad (a name combining small and mothers against decapentaplegic), Janus kinase/signal transducer and activator of transcription, nuclear factor-B, nuclear factor of activated T cells] (12). Increasing lines of evidence suggest that cross talk among intracellular signaling pathways mediate downstream transcriptional responses. Thus, it is important to identify the extracellular ligands, membrane receptors, and signal transduction pathways associated with gonadal development, as well as the transcription factors.
To date, numerous candidate genes have been identified based on their pattern of expression in the embryonic gonad. Gonadal phenotypes are increasingly recognized in knockout models originally designed to explore gene function in other tissues. In many cases, embryonic lethality precludes analysis of adult reproductive function. Hence, for gene mutations that have pleiotropic effects, the gonadal phenotype must be studied primarily during embryogenesis, or by cell type-specific removal of a genetic locus. Further identification of novel candidate genes will rely on a combination of molecular and genetic tools, including forward and reverse genetics approaches.
A model for gonadal development postulates a series of sequential commitment steps as specific cell types achieve their final differentiated state. A goal in the field is to identify the genetic cascade that programs these events. Some of these steps are cell autonomous, meaning that a cell will pursue a particular fate, relatively independent of its environment. More often, key developmental steps occur via cell-to-cell communication, reflecting direct cellular contacts or responses to paracrine signals. Markers of differentiation include morphological changes, as well as alterations in gene or protein expression. A future challenge is to identify specific targets of key genes in the developmental cascade and to characterize the biochemical events associated with differentiation.
Organogenesis in the Urogenital Ridge
After gastrulation, organogenesis in the mouse embryo starts approximately 8 d post coitum (dpc). The gonad, adrenal gland, kidney, and reproductive tract can be traced to a derivative of the intermediate mesoderm—the urogenital ridge—that forms at embryonic d 9.5. At 10.5 dpc, the genital ridge thickens bilaterally on either side of the dorsal aorta and constitutes an outgrowth of epithelial cells that proliferate at the center of the coelomic cavity and extend along the anterior-posterior axis from the forelimb bud to the hindlimb bud. The urogenital ridge is tethered to the peritoneal cavity by mesentery and comprises two juxtaposed tissues: the gonad rudiment is located medially and the mesonephros, which contains dual Wolffian and Müllerian ducts, is more dorsal (Fig. 1, A and B). The metanephric kidney is an outgrowth of the caudal mesonephros and is visible by 12.5 dpc (Fig. 1B). The adreno-gonad primordium arises between the gonad and mesonephros on the dorsal side and buds anteriorly at 12.5 dpc to form the adrenal gland. In both sexes, the adrenal gland and kidney ascend toward the abdominal region. In contrast, the final position of the gonad depends on its differentiation into an ovary or testis, and on the interdependent maturation of the sex-specific Müllerian or Wolffian ducts. In the male, owing the action of testosterone, the proximal Wolffian duct is virilized to form the epididymis, vas deferens, and seminal vesicle. Regression of the distal Müllerian duct is mediated by anti-Müllerian hormone (AMH, or Müllerian inhibitory substance, MIS) (13). Another peptide hormone, insulin-like 3 (Insl-3) mediates testicular descent (13, 14). Persistent Müllerian duct syndrome (PMDS) in males is caused by loss of MIS, MIS type I, or type II receptor function (15, 16, 17). The absence of testosterone and MIS in females allows regression of the Wolffian duct and development of the Müllerian duct into oviduct, uterus, and upper vagina.
FIG. 1. At 11.5 dpc (A), the bipotential urogenital ridge (large arrow) flanks the dorsal aorta (small arrow) along the anterior-posterior axis in between the forelimb bud (FL) and hindlimb bud (HL). The gonad is closer to the midline, and the mesonephros develops dorso-lateral to the gonad. At 11.5 dpc, there are approximately 18 tail somites (ts) from the tip of the tail to the posterior end of the hindlimb bud. B, At 12.5 dpc, the male gonad shows distinct morphologic features including a prominent coelomic blood vessel (arrow). G, Gonad; M, mesonephros; K, kidney. C, By 13.5 dpc, the male gonad (left) is approximately twice the size of the female (right) due to increased cellular proliferation. D, Sexual dimorphism between the male (left) and female (right) gonad is further evident by 14.5 dpc (scale bars, 500 μm).
Genetic Program of the Bipotential Gonad
The gonad primordium is morphologically indistinguishable from 10.0 through 11.5 dpc in males and females. The testis cords become visible by 12.0 dpc and reflect the coalescence of Sertoli and germ cells that are surrounded by a layer of thin peritubular myoid cells. In males, the gonad begins to enlarge visibly at about 13.5 dpc, as a consequence of proliferation of germ cells and precursor somatic cells. The ovary, on the other hand, is seemingly more quiescent during this time, but female-specific markers provide evidence that ovarian differentiation is underway (18, 19).
Before expression of the male determining gene, Sry at 10.5 dpc, a number of factors act in concert to specify the urogenital ridge in both sexes (Fig. 2). Moreover, because the urogenital ridge is the primordium for the gonad, adrenal, kidney, and reproductive tract, multiple organs are often affected by mutations of these genes.
FIG. 2. Summary of genetic pathways involved in gonadal development. The bipotential gonad expresses genes such as Wt1, Sf1, Emx2, M33, Lhx9, Pod1, and Dmrt1. Subsequently, expression of the insulin receptor family Ir, Irr, and Igf1r; GATA4/Fog2; Wt1; and Sf1 are thought to promote the expression of Sry in the male. Sox9 and Sox8 are downstream of Sry. Sf1 regulates Amh activation in Sertoli cells and steroidogenesis in Leydig cells. In the male, Dax1 may repress Sf1 in a dose-dependent manner, modulating the transactivation of Amh and genes necessary for steroid hormone biosynthesis. Dax1 also supports the normal formation of testis cords. Additional factors such as Arx, Dhh, Pdgfr-, Fgf9, and FgfR2 are involved in Leydig and Sertoli cell differentiation. A description of the cellular events downstream of Sry and thus specific to male development is outlined in the center box. PGC, Primordial germ cell; PTM, peritubular myoid cell. Female differentiation requires the activity of Wnt4a and repression of Sox9. Other key factors include Fst, Bmp2, and Dax1.
Wt1 (Wilms’ tumor-1) encodes a zinc finger transcription factor originally identified as a tumor suppressor gene. Loss-of-function mutations cause three distinct pediatric syndromes that display urogenital defects affecting male development: WAGR (Wilms’ tumor, aniridia, genitourinary abnormality, mental retardation), Denys-Drash syndrome, and Frasier syndrome. The Wt1 homozygous knockout animal does not form kidney, adrenal, or gonad (20, 21). In the gonad, the expression of Wt1 at embryonic d 9.5 suggests a role in specifying the coelomic epithelial cells in the developing urogenital ridge and ensuring their survival (22). Two major isoforms have relevance to the developing urogenital organs: one alternatively spliced variant contains amino acids KTS (lysine-threonine-serine) (+KTS isoform), and the other lacks KTS (–KTS isoform). The additional amino acids lend flexibility to the protein between the third and fourth zinc finger and thereby diminish its DNA binding property. Both isoforms appear to have nonredundant functions in kidney and gonad development but not in the adrenal (23). In animals with genetically engineered ablation of the –KTS isoform, apoptosis was observed in streak gonads. Sry expression is reduced in the gonads of animals lacking the +KTS splice variant. Loss of either splice variant impairs testis descent.
Wt1 functions upstream of two orphan nuclear receptors Sf1 (steroidogenic factor 1) and Dax1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, X chromosome) (11). Wt1 and Sf1 synergize to enhance transcription of Amh, whereas in vitro experiments demonstrate this interaction is repressed by Dax1 (10). Sf1 also regulates the expression of multiple genes involved in male differentiation, steroidogenesis, and reproduction through Sf1-responsive DNA elements in target gene promoters [Ref.24 ; and see review in this issue by Hammer et al. (25)]. Sf1 stimulates Dax1 transcription, and the two receptors are coexpressed in the adrenal, gonad, pituitary gonadotropes, and ventromedial hypothalamus (26). Dax1 interacts directly with Sf1 and functions as a transcriptional repressor of Sf1-regulated genes (27). In Sf1 null mutants, neither XX nor XY animals form the adrenal or gonad. In the absence of Sf1, cells in both tissue rudiments undergo apoptosis, indicating that Sf1 is necessary for survival of early progenitors of the adrenal and gonad (28). At 11.5 dpc, Sf1 colocalizes with proliferating cells, suggesting that it directly or indirectly stimulates cell proliferation (29). Dax1-null males possess gonadal defects in testis cord morphogenesis, peritubular myoid cell proliferation, and spermatogenesis (30, 31). Hence, Sf1 and Dax1 are independently important for normal male gonadal differentiation.
Genes that primarily affect male gonadal differentiation or, have a male-specific expression pattern before distinct morphological changes, continue to expand the network of molecular pathways involved in testis development. Autosomal recessive male-to-female sex reversal (XY male bearing female gonads and feminized external genitalia) has been described in several mouse knockout models. M33 (polycomb homolog) is involved in chromatin remodeling, yet its precise molecular role in gonadal differentiation is unknown (9). The LIM-domain containing transcription factor Lhx9 (Lim homeobox) is required for full Sf1 expression (11, 32). In contrast to the Sf1 null animal, lack of testis development was not attributed to apoptosis in either Lhx9 or M33 mutant mice. Pod1 (podocyte) is a basic helix-loop-helix transcription factor with a primary role in glomerular visceral epithelial cell (podocyte) differentiation and branching morphogenesis in the kidney and lung (33). In Pod1 knockout mice, the gonads of both sexes are hypoplastic (34). Expression of Pod1 in the male gonad was found mainly in the interstitium: peritubular myoid cells, pericytes associated with endothelium, and fetal Leydig cells. Defective coelomic vessel formation and attenuation of migrating endothelial cells from the mesonephros compromised vascularization in Pod1 knockout male gonads. Although Sertoli development was not affected, there appeared to be expansion of the Sf1-positive Leydig cell population. Dmrt1 (doublesex- and mab-related transcription factor) is homologous to genes in Drosophila and Caenorhabditis elegans, and null mutation causes dysmorphic testes in mice (35). Maestro (Mro), protease nexin-1 (Pn-1), and vanin-1 (Vn-1) were identified based on a sexually dimorphic expression pattern specific to the XY bipotential gonad (36, 37, 38). Later in development, maestro remains expressed in Sertoli and XY germ cells, whereas protease nexin-1 and vanin-1 are Sertoli restricted. Whether any of these newly described factors specify precursor populations, analogous to the role of Sry-expressing Sertoli precursor cells, remains to be tested. The extent to which function can be attributed to a gene product relies on the phenotype of null mutation models in which are affected on all tissues where the gene of interest is expressed. Only recently have tissue-specific mutagenesis studies, for example with Sf1 and Sox9 [Sry related, HMG (high-mobility group) box], begun to elucidate spatial requirements attributed to developmentally regulated genes (39, 40, 41).
Supporting Cells: The Embryonic Testis Cord
The Sertoli cell is positioned at the base of the testis cord creating the perimeter of the tubule. Closely apposed is the elongated cell layer of peritubular myoid cells circumferentially surrounding the nascent cord. Testis cord morphogenesis is apparent in the male as early as embryonic d 12.0. The testis cords pattern the developing gonad and are the embryologic foundation for the adult seminiferous tubule. Sertoli precursor cells were identified at the coelomic surface (ventro-medial aspect) using dye tracking experiments that labeled dividing cells (42). Sertoli cells and granulosa cells may originate from the same precursor (43). In XX-XY chimeric studies, Sertoli cells were the only cell type that required Sry expression in a cell autonomous manner, whereas XX cells were preferentially excluded from the Sertoli population (44).
Sertoli cells, the cell lineage in which Sry is solely expressed in the male gonad, are thought to direct the differentiation of other cell types (45). For example, fetal Leydig cell differentiation depends on paracrine signaling through the extracellular protein Desert Hedgehog, which is secreted by Sertoli cells (46). Endothelial cells originating from the mesonephros are induced by a chemoattractant released by the male gonad (47). Two factors that can induce mesonephric cell migration are fibroblast growth factor (FGF) 9 and MIS, both of which are produced by Sertoli cells (48, 49). Sertoli cells also secrete a postulated factor(s) that induces mitotic arrest of germ cells (50). Unlike the female gonad, germ cells in the developing testis undergo mitotic arrest, being held as diploid progenitors until later in spermatogenesis. There is recent evidence that XY germ cells synthesize paracrine factors, including prostaglandin D2, that induce Sertoli differentiation (51).
Sry expression occurs in a narrow temporal window from 10.5–12.0 dpc. The spatial pattern of Sry expression has been clarified by in situ hybridization analysis timed to tail somite stages. The central region of the gonad exhibits Sry expression first, followed by a rostral to caudal progression along the entire length of the gonad (52). Although Sry belongs to the HMG box containing family of nuclear transcription factors, evidence for downstream target genes remains elusive. A closely related gene, Sox9, is the other definitive male-determining gene identified thus far. Immunohistochemical labeling of genetically marked Sry-positive cells demonstrate that Sry and Sox9 expression overlap in cells of the Sertoli lineage (53). As the Sry signal diminishes at 12.5 dpc, Sox9 expression increases in the male and reaches a peak at 14.5 dpc. Sox9, which is weakly expressed in the undifferentiated gonad, is down-regulated in the female. Transgenic expression of Sox9 on an XX genetic background is sufficient to cause female-to-male sex reversal (54). Heterozygous human SOX9 mutations cause campomelic dysplasia, a severe skeletal disorder with defective cartilage development (55, 56). Many of these male patients also have gonadal dysgenesis. Heterozygous mice haploinsufficient for Sox9 die perinatally due to skeletal malformations (57). An ingenious strategy was designed to transmit the knockout allele through the germline of otherwise wild-type male and female parents by the use of spermatocyte- and oocyte-specific promoters, respectively, enabling the zygote to obtain both knockout alleles (41). These experiments confirm that Sox9 is necessary for male gonadal development. Notably, Sox9 null embryos have elevated Sry expression, suggesting a negative feedback regulatory loop that down-regulates Sry in the mouse. Experimental evidence using mouse strains with a variety of Sry alleles supports the assertion that the precise timing, duration, threshold transcript levels, and protein isoform half-life, influence downstream events dependent on Sry expression (58, 59). Sox8, another member of the Sox HMG box containing transcription factor gene family, is also active in male development (60). It is possible that Sry, Sox9, and Sox8 have partially redundant functions.
GATA4 and Fog2 (friend of GATA) are important for cardiac morphogenesis but also influence gonadal development. A GATA4 knock-in mutation, which prevents nuclear association of GATA4 and Fog2, eliminates expression of the male differentiation markers Sox9 and Amh. Fog2 null mice have decreased Sry expression, loss of Sox9, Amh, and Desert hedgehog (Dhh), but persistence of the female marker Wnt4 (61). The GATA4 knock-in and Fog2 null mice retain Sf1 and Wt1 expression, suggesting a hierarchical network. Similarly, Ir, Irr, Igf1r (insulin receptor, insulin-related receptor, Igf1 receptor) triple mutants have low Sry and Sox9 expression and exhibit male-to-female sex reversal, thus implicating the insulin signaling pathway in male differentiation (62).
Anti-Müllerian hormone (AMH) or MIS is a key peptide hormone produced by Sertoli cells. It mediates the regression of the Müllerian duct, which would otherwise form female reproductive tract structures. In genetic males, PMDS is due to loss of MIS or cognate receptors present in mesenchymal cells of the Müllerian duct. PMDS is an example of male pseudohermaphroditism whereby males with testicular tissue appear feminized externally due to failure of testis descent caused by the presence of uterus and oviduct. Müllerian regression involves activation of a ?-catenin-dependent pathway in a rostral-to-caudal manner (63). A number of factors are known to regulate Amh transactivation. These include Sf1, WT1, GATA4, Sox9, and Sox8 (10, 64, 65, 66). MIS may have other functions that include mesonephric cell migration and vascularization in the male gonad, and germ cell loss in the ovary (49, 67).
Proliferation of pre-Sertoli cells is an important event in male development (29). By 13.5 dpc, the male gonad is twice the size of the female (see Fig. 1, C and D). One factor linked to proliferation is Fgf9. Fgf9 null mice show varying degrees of male-to-female sex reversal (48). A number of Fgf receptors have been identified and FgfR2 is thought to direct Sertoli cell differentiation perhaps as a result of nuclear translocation, which occurs simultaneously with translocation of Sry and Sox9 (68). A secreted protein, Fgf9, is a candidate male gonad-specific chemoattractant signal that induces migration of cells from the mesonephros into the gonad. Immigrant cells have been identified mainly as endothelial and peritubular myoid cells (47). Migration of these cells is necessary for testis cord morphogenesis (69) and induction of Sox9 expression (70). This migration of mesonephric cells does not occur in females presumably because a chemoattractant is absent. Furthermore, once XX germ cells enter meiosis at 13.5 dpc, migration from the mesonephros is inhibited (71).
Hormone Biosynthesis: The Interstitial Space
Once sex determination has occurred, the ensuing phenotypic differentiation depends heavily on the production of steroid hormones. In the male, testosterone stabilizes the Wolffian duct and is converted by 5-reductase to the potent derivative, dihydrotestosterone, which induces virilization of the male external genitalia. In the female, aromatase mediates estradiol synthesis. In some species, aromatase expression is critical for ovarian development; its role in ovary development in mammals is less clear, although aromatase deficiency is associated with transdifferentiation of granulosa cells into Sertoli-like cells (72).
Sf1 is restricted to Leydig cells after 13.5 dpc and mediates expression of several genes encoding enzymes required for testosterone biosynthesis including StAR (steroidogenic acute regulatory protein), Cyp11a1, Cyp17 (cytochrome P450 hydroxylases), and 3?HSD (hydroxysteroid dehydrogenase). The factors required for Leydig cell determination and lineage development are incompletely understood. Moreover, their origin, whether from immigrant mesonephric cells or progenitors within the gonad, remains equivocal. Leydig cell fate appears to rely on paracrine signals. Dhh, a Sertoli-secreted factor, induces expression of Cyp11a1 (side-chain cleavage) in the Leydig cell (46). The Dhh signal is received by the receptor patched (Ptc1), which is expressed on Leydig cells. Dhh is thought to play a crucial role in Leydig cell differentiation and is an example of a paracrine signaling pathway identified between two developing cell types. Genetic analysis has placed Pdgfr- (platelet-derived growth factor receptor-) upstream of Dhh (73). Like Fgf9, Pdgfs induce mesonephric migration, and Pdgfr- is necessary in the gonad for full Cyp11a1 expression. The X-linked gene Arx (Aristaless-related homeobox) also influences Leydig cell development. However, it is expressed mainly in peritubular myoid cells, endothelial cells, and in the epithelium of the tunica albuginea (74).
Female Embryonic Gonadal Development
Relatively few genes have been shown to exhibit a female-specific pattern of gene expression early in gonadal development. To date, no gene has been shown to be a female-determining gene, as defined by conferring complete female-to-male sex reversal when mutated on an XX background, or by conferring male-to-female sex reversal after overexpression on an XY background. The Dax1 gene was initially suggested as a pro-ovarian, or anti-testis, candidate gene because its duplication on an XY background is associated with impaired testis development (75, 76). However, Dax1 loss of function on the XX background does not prevent ovary development (31). Subsequent studies have shown an unexpected role for Dax1 in testis development (77), indicating that its actions are highly dependent on the timing and level of expression.
Although male somatic tissue can survive in the absence of germ cells, the female gonad depends on migratory germ cells to populate the undifferentiated primordium (78). In the female gonad, germ cells begin entering meiosis at 13.5 dpc and initiate a dynamic paracrine relationship with supporting cells destined to become cumulus and granulosa cells (79).
A growing list of genes exhibit an ovary-specific expression pattern. For example, follistatin (Fst) is highly expressed in the developing ovary relative to the testis (19). Follistatin binds members of the activin/TGF?/bone morphogenetic protein (BMP) family. Thus, it may regulate the activity of one or more of these factors. Stra8 (stimulated by retinoic acid) is also selectively expressed in the developing ovary and is thought to play a role in regulation of meiosis (80). Targeted mutagenesis and transgenic overexpression experiments will clarify the functions of these factors.
There has been postulated a "Z factor" that could act to suppress pro-testis events carried out by autosomal or X-linked genes in both XY and XX backgrounds (81). The Z factor would act downstream of Sry in genetic males. Therefore, the Z factor would be repressed by Sry in the male, and independent of Sry on an XX genetic background. Loss of a Z factor would be sex-reversing on XX (female-to-male), but gain-of-function on XY may or may not exhibit sex reversal (male-to-female) depending on whether the Z factor can override the downstream events of Sry. One candidate for the Z factor is Wnt4 (Wingless-related integration site). Female Wnt4 null mice develop virilizing gonads and Wolffian derivatives (82). This gonadal phenotype reflects the action of Wnt4 to induce the migration of steroidogenic precursors of the adreno-gonad primordium into the anterior region of the gonad (83). Hence, misdirected adrenal steroidogenic cells become localized in the XX Wnt4 knockout gonad. Secondly, Wnt4 is down-regulated in males, whereas its expression remains strong in females where it prevents coelomic vascularization (a male feature, see Fig. 1B) through a molecular pathway that involves Fst and Bmp2 (19). By inhibiting vascularization, Wnt4 may prevent the proliferative growth expansion characteristic of male development. Thus, the homozygous knockout female gonad appears somewhat more male-like in shape and size. Externally, however, homozygous knockouts exhibit female genitalia. In humans, a loss-of-function mutation in WNT4 caused Mayer-Rokitansky-Kuster-Hauser syndrome, which is characterized by defective development of Müllerian derivatives (84). Duplication of a chromosomal region containing WNT4 was associated with a case of human XY sex reversal (85). However, overexpression of Wnt4 on an XY background does not lead to XY sex reversal in the mouse (83, 86). Thus, the major roles of Wnt4 in the female appear to include suppression of androgen-producing interstitial cells, inhibition of testis-like vascularization, and support of Müllerian derivatives. Wnt4 is also essential for kidney (87), mammary gland (88), and pituitary development (89), as well as normal functioning of the adrenal cortex (90).
A dominant insertional mutation led to identification of a female-to-male sex reversal phenotype in the Odsex (Ocular degeneration with sex reversal) mouse (91). The Odsex mouse was initially thought to lack a repressor for Sox9, allowing unabated Sox9 expression ectopically in genetic females. Therefore, one of the functions of a Z factor could be to suppress Sox9 action in the female gonad. However, it is also possible that regulatory elements contained in the inserted transgene promote activation of Sox9.
Nucleo-cytoplasmic shuttling of transcription factors may also provide an important regulatory step in sex determination. Organ culture experiments using the nuclear export blocker, leptomycin B, resulted in male-characteristic Sox9 and MIS expression in XX gonad organ cultures (92). Thus, growth factors may be able to modulate transcriptional control by altering the location as well as the activity of various transcription factors.
Central Questions and Future Directions
The molecular pathways directing cellular differentiation can be traced back to cells of pluripotential origin. Thus, development must occur through a series of commitment steps driven by cell-to-cell contacts and paracrine interactions. In some cases, committed cells initiate a cell-autonomous program, as perhaps illustrated by Sry and Sox9 actions in Sertoli cells. In other cases, differentiation is driven by secreted factors as illustrated by Dhh, Wnt4, and Fgf9. These cell autonomous and paracrine pathways are not mutually exclusive, and it is likely that most commitment steps integrate a series of internal and external signals. The sex determination pathway provides a unique paradigm for considering these events because the commitment steps lead to distinct cell types within a narrow time frame.
Despite impressive progress in this field, a number of challenging questions remain. What factors initiate Sry expression and what are its cellular targets? Are Sry and Sox9 expression linked directly or do they reflect parallel, partially redundant pathways? Given that Sox9 is sufficient to induce Sertoli cell development, what are its targets and how does it induce a network of genes they convey morphological changes characteristic of the testis? What are the genetic and molecular events that dictate ovary development? Although it is known that germ cells interact actively with somatic cells, what are the molecular and cellular events that control germ cell replication and entry into meiosis? How do meiotic germ cells foster ovary development? Implicit in these questions is the critical feature of timing during gonadal development. Once the Sertoli program is initiated, the developing testis induces mitotic arrest of germ cells. On the other hand, if Sertoli development is delayed, as occurs with various Sry alleles and mouse genetic backgrounds, germ cells begin to enter meiosis, and this step may further impair testis development, leading to mixed testis/ovary tissue or gonadal dysgenesis. Thus, future studies need to carefully assess pathways that control the timing and level of expression of regulatory factors.
Further understanding of these molecular and cellular functions in the gonad will provide examples of paradigms used to control cellular differentiation in other tissues. This knowledge will also be useful for the characterization of intersex cases and perhaps additional causes of idiopathic infertility. Parallel investigation of human mutations and animal models can accelerate progress in this field by identifying candidate genes and clarifying their roles in development.
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Address all correspondence and requests for reprints to: J. Larry Jameson, Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611. E-mail: ljameson@northwestern.edu.
Abstract
The embryonic gonad is undifferentiated in males and females until a critical stage when the sex chromosomes dictate its development as a testis or ovary. This binary developmental process provides a unique opportunity to delineate the molecular pathways that lead to distinctly different tissues. The testis comprises three main cell types: Sertoli cells, Leydig cells, and germ cells. The Sertoli cells and germ cells reside in seminiferous tubules where spermatogenesis occurs. The Leydig cells populate the interstitial compartment and produce testosterone. The ovary also comprises three main cell types: granulosa cells, theca cells, and oocytes. The oocytes are surrounded by granulosa and theca cells in follicles that grow and differentiate during characteristic reproductive cycles. In this review, we summarize the molecular pathways that regulate the distinct differentiation of these cell types in the developing testis and ovary. In particular, we focus on the transcription factors that initiate these cascades. Although most of the early insights into the sex determination pathway were based on human mutations, targeted mutagenesis in mouse models has revealed key roles for genes not anticipated to regulate gonadal development. Defining these molecular pathways provides the foundation for understanding this critical developmental event and provides new insight into the causes of gonadal dysgenesis.
LIKE MOST ORGAN systems, mammalian gonadal development involves a complex interplay of multiple cell types, and it occurs during a relatively narrow time window. Thus, it is important to understand the temporal pattern of gene expression as well as the spatial relationships of the developing tissues. For these reasons, most recent studies have used the mouse as a model for examining gonadal development because it is amenable to genetic manipulation as well as detailed histologic and biochemical characterization. During the transition from an undifferentiated gonad to a testis or ovary, the female and male gonad each display characteristic morphological features and patterns of gene expression (1). The coordinated differentiation of the embryonic urogenital ridge, from the bipotential state to sexual dimorphism, provides an opportunity to identify the key factors and commitment steps that underlie gonadal differentiation (2).
In humans, the functional significance of some of these molecules is evident from mutations that impair gonadal development and reproduction (3, 4, 5). For example, deletions of the Y chromosomal gene, SRY (sex-determining region on the Y chromosome) cause XY male-to-female sex reversal, whereas SRY translocations to the X chromosome lead to XX female-to-male sex reversal (6). The role of Sry as a candidate testis-determining gene was confirmed by demonstrating testis development after transgenic expression of Sry in XX mice (7). Thus, by using animal models, it is possible to explore genetic pathways in greater detail and elucidate disease pathogenesis.
Many of the gonadal development factors described to date are known to act at the transcriptional level. For the most part, their functions are incompletely understood. Based on homology to other transcription factors, some affect DNA bending (8) or modulate chromatin remodeling (9). Others form interactive complexes that activate transcription (10) or have a role in specifying progenitor cell types (11). Inhibition of gene expression is equally likely to be important as a means to dictate cell fate, but less is known about potential transcriptional repressors. The functional interaction between extracellular ligand molecules and nuclear transcription factors also merits emphasis. For example, extracellular signals can induce transcription factor release and translocation to the nucleus [i.e. ?-catenin/lymphoid-enhancing factor, Smad (a name combining small and mothers against decapentaplegic), Janus kinase/signal transducer and activator of transcription, nuclear factor-B, nuclear factor of activated T cells] (12). Increasing lines of evidence suggest that cross talk among intracellular signaling pathways mediate downstream transcriptional responses. Thus, it is important to identify the extracellular ligands, membrane receptors, and signal transduction pathways associated with gonadal development, as well as the transcription factors.
To date, numerous candidate genes have been identified based on their pattern of expression in the embryonic gonad. Gonadal phenotypes are increasingly recognized in knockout models originally designed to explore gene function in other tissues. In many cases, embryonic lethality precludes analysis of adult reproductive function. Hence, for gene mutations that have pleiotropic effects, the gonadal phenotype must be studied primarily during embryogenesis, or by cell type-specific removal of a genetic locus. Further identification of novel candidate genes will rely on a combination of molecular and genetic tools, including forward and reverse genetics approaches.
A model for gonadal development postulates a series of sequential commitment steps as specific cell types achieve their final differentiated state. A goal in the field is to identify the genetic cascade that programs these events. Some of these steps are cell autonomous, meaning that a cell will pursue a particular fate, relatively independent of its environment. More often, key developmental steps occur via cell-to-cell communication, reflecting direct cellular contacts or responses to paracrine signals. Markers of differentiation include morphological changes, as well as alterations in gene or protein expression. A future challenge is to identify specific targets of key genes in the developmental cascade and to characterize the biochemical events associated with differentiation.
Organogenesis in the Urogenital Ridge
After gastrulation, organogenesis in the mouse embryo starts approximately 8 d post coitum (dpc). The gonad, adrenal gland, kidney, and reproductive tract can be traced to a derivative of the intermediate mesoderm—the urogenital ridge—that forms at embryonic d 9.5. At 10.5 dpc, the genital ridge thickens bilaterally on either side of the dorsal aorta and constitutes an outgrowth of epithelial cells that proliferate at the center of the coelomic cavity and extend along the anterior-posterior axis from the forelimb bud to the hindlimb bud. The urogenital ridge is tethered to the peritoneal cavity by mesentery and comprises two juxtaposed tissues: the gonad rudiment is located medially and the mesonephros, which contains dual Wolffian and Müllerian ducts, is more dorsal (Fig. 1, A and B). The metanephric kidney is an outgrowth of the caudal mesonephros and is visible by 12.5 dpc (Fig. 1B). The adreno-gonad primordium arises between the gonad and mesonephros on the dorsal side and buds anteriorly at 12.5 dpc to form the adrenal gland. In both sexes, the adrenal gland and kidney ascend toward the abdominal region. In contrast, the final position of the gonad depends on its differentiation into an ovary or testis, and on the interdependent maturation of the sex-specific Müllerian or Wolffian ducts. In the male, owing the action of testosterone, the proximal Wolffian duct is virilized to form the epididymis, vas deferens, and seminal vesicle. Regression of the distal Müllerian duct is mediated by anti-Müllerian hormone (AMH, or Müllerian inhibitory substance, MIS) (13). Another peptide hormone, insulin-like 3 (Insl-3) mediates testicular descent (13, 14). Persistent Müllerian duct syndrome (PMDS) in males is caused by loss of MIS, MIS type I, or type II receptor function (15, 16, 17). The absence of testosterone and MIS in females allows regression of the Wolffian duct and development of the Müllerian duct into oviduct, uterus, and upper vagina.
FIG. 1. At 11.5 dpc (A), the bipotential urogenital ridge (large arrow) flanks the dorsal aorta (small arrow) along the anterior-posterior axis in between the forelimb bud (FL) and hindlimb bud (HL). The gonad is closer to the midline, and the mesonephros develops dorso-lateral to the gonad. At 11.5 dpc, there are approximately 18 tail somites (ts) from the tip of the tail to the posterior end of the hindlimb bud. B, At 12.5 dpc, the male gonad shows distinct morphologic features including a prominent coelomic blood vessel (arrow). G, Gonad; M, mesonephros; K, kidney. C, By 13.5 dpc, the male gonad (left) is approximately twice the size of the female (right) due to increased cellular proliferation. D, Sexual dimorphism between the male (left) and female (right) gonad is further evident by 14.5 dpc (scale bars, 500 μm).
Genetic Program of the Bipotential Gonad
The gonad primordium is morphologically indistinguishable from 10.0 through 11.5 dpc in males and females. The testis cords become visible by 12.0 dpc and reflect the coalescence of Sertoli and germ cells that are surrounded by a layer of thin peritubular myoid cells. In males, the gonad begins to enlarge visibly at about 13.5 dpc, as a consequence of proliferation of germ cells and precursor somatic cells. The ovary, on the other hand, is seemingly more quiescent during this time, but female-specific markers provide evidence that ovarian differentiation is underway (18, 19).
Before expression of the male determining gene, Sry at 10.5 dpc, a number of factors act in concert to specify the urogenital ridge in both sexes (Fig. 2). Moreover, because the urogenital ridge is the primordium for the gonad, adrenal, kidney, and reproductive tract, multiple organs are often affected by mutations of these genes.
FIG. 2. Summary of genetic pathways involved in gonadal development. The bipotential gonad expresses genes such as Wt1, Sf1, Emx2, M33, Lhx9, Pod1, and Dmrt1. Subsequently, expression of the insulin receptor family Ir, Irr, and Igf1r; GATA4/Fog2; Wt1; and Sf1 are thought to promote the expression of Sry in the male. Sox9 and Sox8 are downstream of Sry. Sf1 regulates Amh activation in Sertoli cells and steroidogenesis in Leydig cells. In the male, Dax1 may repress Sf1 in a dose-dependent manner, modulating the transactivation of Amh and genes necessary for steroid hormone biosynthesis. Dax1 also supports the normal formation of testis cords. Additional factors such as Arx, Dhh, Pdgfr-, Fgf9, and FgfR2 are involved in Leydig and Sertoli cell differentiation. A description of the cellular events downstream of Sry and thus specific to male development is outlined in the center box. PGC, Primordial germ cell; PTM, peritubular myoid cell. Female differentiation requires the activity of Wnt4a and repression of Sox9. Other key factors include Fst, Bmp2, and Dax1.
Wt1 (Wilms’ tumor-1) encodes a zinc finger transcription factor originally identified as a tumor suppressor gene. Loss-of-function mutations cause three distinct pediatric syndromes that display urogenital defects affecting male development: WAGR (Wilms’ tumor, aniridia, genitourinary abnormality, mental retardation), Denys-Drash syndrome, and Frasier syndrome. The Wt1 homozygous knockout animal does not form kidney, adrenal, or gonad (20, 21). In the gonad, the expression of Wt1 at embryonic d 9.5 suggests a role in specifying the coelomic epithelial cells in the developing urogenital ridge and ensuring their survival (22). Two major isoforms have relevance to the developing urogenital organs: one alternatively spliced variant contains amino acids KTS (lysine-threonine-serine) (+KTS isoform), and the other lacks KTS (–KTS isoform). The additional amino acids lend flexibility to the protein between the third and fourth zinc finger and thereby diminish its DNA binding property. Both isoforms appear to have nonredundant functions in kidney and gonad development but not in the adrenal (23). In animals with genetically engineered ablation of the –KTS isoform, apoptosis was observed in streak gonads. Sry expression is reduced in the gonads of animals lacking the +KTS splice variant. Loss of either splice variant impairs testis descent.
Wt1 functions upstream of two orphan nuclear receptors Sf1 (steroidogenic factor 1) and Dax1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, X chromosome) (11). Wt1 and Sf1 synergize to enhance transcription of Amh, whereas in vitro experiments demonstrate this interaction is repressed by Dax1 (10). Sf1 also regulates the expression of multiple genes involved in male differentiation, steroidogenesis, and reproduction through Sf1-responsive DNA elements in target gene promoters [Ref.24 ; and see review in this issue by Hammer et al. (25)]. Sf1 stimulates Dax1 transcription, and the two receptors are coexpressed in the adrenal, gonad, pituitary gonadotropes, and ventromedial hypothalamus (26). Dax1 interacts directly with Sf1 and functions as a transcriptional repressor of Sf1-regulated genes (27). In Sf1 null mutants, neither XX nor XY animals form the adrenal or gonad. In the absence of Sf1, cells in both tissue rudiments undergo apoptosis, indicating that Sf1 is necessary for survival of early progenitors of the adrenal and gonad (28). At 11.5 dpc, Sf1 colocalizes with proliferating cells, suggesting that it directly or indirectly stimulates cell proliferation (29). Dax1-null males possess gonadal defects in testis cord morphogenesis, peritubular myoid cell proliferation, and spermatogenesis (30, 31). Hence, Sf1 and Dax1 are independently important for normal male gonadal differentiation.
Genes that primarily affect male gonadal differentiation or, have a male-specific expression pattern before distinct morphological changes, continue to expand the network of molecular pathways involved in testis development. Autosomal recessive male-to-female sex reversal (XY male bearing female gonads and feminized external genitalia) has been described in several mouse knockout models. M33 (polycomb homolog) is involved in chromatin remodeling, yet its precise molecular role in gonadal differentiation is unknown (9). The LIM-domain containing transcription factor Lhx9 (Lim homeobox) is required for full Sf1 expression (11, 32). In contrast to the Sf1 null animal, lack of testis development was not attributed to apoptosis in either Lhx9 or M33 mutant mice. Pod1 (podocyte) is a basic helix-loop-helix transcription factor with a primary role in glomerular visceral epithelial cell (podocyte) differentiation and branching morphogenesis in the kidney and lung (33). In Pod1 knockout mice, the gonads of both sexes are hypoplastic (34). Expression of Pod1 in the male gonad was found mainly in the interstitium: peritubular myoid cells, pericytes associated with endothelium, and fetal Leydig cells. Defective coelomic vessel formation and attenuation of migrating endothelial cells from the mesonephros compromised vascularization in Pod1 knockout male gonads. Although Sertoli development was not affected, there appeared to be expansion of the Sf1-positive Leydig cell population. Dmrt1 (doublesex- and mab-related transcription factor) is homologous to genes in Drosophila and Caenorhabditis elegans, and null mutation causes dysmorphic testes in mice (35). Maestro (Mro), protease nexin-1 (Pn-1), and vanin-1 (Vn-1) were identified based on a sexually dimorphic expression pattern specific to the XY bipotential gonad (36, 37, 38). Later in development, maestro remains expressed in Sertoli and XY germ cells, whereas protease nexin-1 and vanin-1 are Sertoli restricted. Whether any of these newly described factors specify precursor populations, analogous to the role of Sry-expressing Sertoli precursor cells, remains to be tested. The extent to which function can be attributed to a gene product relies on the phenotype of null mutation models in which are affected on all tissues where the gene of interest is expressed. Only recently have tissue-specific mutagenesis studies, for example with Sf1 and Sox9 [Sry related, HMG (high-mobility group) box], begun to elucidate spatial requirements attributed to developmentally regulated genes (39, 40, 41).
Supporting Cells: The Embryonic Testis Cord
The Sertoli cell is positioned at the base of the testis cord creating the perimeter of the tubule. Closely apposed is the elongated cell layer of peritubular myoid cells circumferentially surrounding the nascent cord. Testis cord morphogenesis is apparent in the male as early as embryonic d 12.0. The testis cords pattern the developing gonad and are the embryologic foundation for the adult seminiferous tubule. Sertoli precursor cells were identified at the coelomic surface (ventro-medial aspect) using dye tracking experiments that labeled dividing cells (42). Sertoli cells and granulosa cells may originate from the same precursor (43). In XX-XY chimeric studies, Sertoli cells were the only cell type that required Sry expression in a cell autonomous manner, whereas XX cells were preferentially excluded from the Sertoli population (44).
Sertoli cells, the cell lineage in which Sry is solely expressed in the male gonad, are thought to direct the differentiation of other cell types (45). For example, fetal Leydig cell differentiation depends on paracrine signaling through the extracellular protein Desert Hedgehog, which is secreted by Sertoli cells (46). Endothelial cells originating from the mesonephros are induced by a chemoattractant released by the male gonad (47). Two factors that can induce mesonephric cell migration are fibroblast growth factor (FGF) 9 and MIS, both of which are produced by Sertoli cells (48, 49). Sertoli cells also secrete a postulated factor(s) that induces mitotic arrest of germ cells (50). Unlike the female gonad, germ cells in the developing testis undergo mitotic arrest, being held as diploid progenitors until later in spermatogenesis. There is recent evidence that XY germ cells synthesize paracrine factors, including prostaglandin D2, that induce Sertoli differentiation (51).
Sry expression occurs in a narrow temporal window from 10.5–12.0 dpc. The spatial pattern of Sry expression has been clarified by in situ hybridization analysis timed to tail somite stages. The central region of the gonad exhibits Sry expression first, followed by a rostral to caudal progression along the entire length of the gonad (52). Although Sry belongs to the HMG box containing family of nuclear transcription factors, evidence for downstream target genes remains elusive. A closely related gene, Sox9, is the other definitive male-determining gene identified thus far. Immunohistochemical labeling of genetically marked Sry-positive cells demonstrate that Sry and Sox9 expression overlap in cells of the Sertoli lineage (53). As the Sry signal diminishes at 12.5 dpc, Sox9 expression increases in the male and reaches a peak at 14.5 dpc. Sox9, which is weakly expressed in the undifferentiated gonad, is down-regulated in the female. Transgenic expression of Sox9 on an XX genetic background is sufficient to cause female-to-male sex reversal (54). Heterozygous human SOX9 mutations cause campomelic dysplasia, a severe skeletal disorder with defective cartilage development (55, 56). Many of these male patients also have gonadal dysgenesis. Heterozygous mice haploinsufficient for Sox9 die perinatally due to skeletal malformations (57). An ingenious strategy was designed to transmit the knockout allele through the germline of otherwise wild-type male and female parents by the use of spermatocyte- and oocyte-specific promoters, respectively, enabling the zygote to obtain both knockout alleles (41). These experiments confirm that Sox9 is necessary for male gonadal development. Notably, Sox9 null embryos have elevated Sry expression, suggesting a negative feedback regulatory loop that down-regulates Sry in the mouse. Experimental evidence using mouse strains with a variety of Sry alleles supports the assertion that the precise timing, duration, threshold transcript levels, and protein isoform half-life, influence downstream events dependent on Sry expression (58, 59). Sox8, another member of the Sox HMG box containing transcription factor gene family, is also active in male development (60). It is possible that Sry, Sox9, and Sox8 have partially redundant functions.
GATA4 and Fog2 (friend of GATA) are important for cardiac morphogenesis but also influence gonadal development. A GATA4 knock-in mutation, which prevents nuclear association of GATA4 and Fog2, eliminates expression of the male differentiation markers Sox9 and Amh. Fog2 null mice have decreased Sry expression, loss of Sox9, Amh, and Desert hedgehog (Dhh), but persistence of the female marker Wnt4 (61). The GATA4 knock-in and Fog2 null mice retain Sf1 and Wt1 expression, suggesting a hierarchical network. Similarly, Ir, Irr, Igf1r (insulin receptor, insulin-related receptor, Igf1 receptor) triple mutants have low Sry and Sox9 expression and exhibit male-to-female sex reversal, thus implicating the insulin signaling pathway in male differentiation (62).
Anti-Müllerian hormone (AMH) or MIS is a key peptide hormone produced by Sertoli cells. It mediates the regression of the Müllerian duct, which would otherwise form female reproductive tract structures. In genetic males, PMDS is due to loss of MIS or cognate receptors present in mesenchymal cells of the Müllerian duct. PMDS is an example of male pseudohermaphroditism whereby males with testicular tissue appear feminized externally due to failure of testis descent caused by the presence of uterus and oviduct. Müllerian regression involves activation of a ?-catenin-dependent pathway in a rostral-to-caudal manner (63). A number of factors are known to regulate Amh transactivation. These include Sf1, WT1, GATA4, Sox9, and Sox8 (10, 64, 65, 66). MIS may have other functions that include mesonephric cell migration and vascularization in the male gonad, and germ cell loss in the ovary (49, 67).
Proliferation of pre-Sertoli cells is an important event in male development (29). By 13.5 dpc, the male gonad is twice the size of the female (see Fig. 1, C and D). One factor linked to proliferation is Fgf9. Fgf9 null mice show varying degrees of male-to-female sex reversal (48). A number of Fgf receptors have been identified and FgfR2 is thought to direct Sertoli cell differentiation perhaps as a result of nuclear translocation, which occurs simultaneously with translocation of Sry and Sox9 (68). A secreted protein, Fgf9, is a candidate male gonad-specific chemoattractant signal that induces migration of cells from the mesonephros into the gonad. Immigrant cells have been identified mainly as endothelial and peritubular myoid cells (47). Migration of these cells is necessary for testis cord morphogenesis (69) and induction of Sox9 expression (70). This migration of mesonephric cells does not occur in females presumably because a chemoattractant is absent. Furthermore, once XX germ cells enter meiosis at 13.5 dpc, migration from the mesonephros is inhibited (71).
Hormone Biosynthesis: The Interstitial Space
Once sex determination has occurred, the ensuing phenotypic differentiation depends heavily on the production of steroid hormones. In the male, testosterone stabilizes the Wolffian duct and is converted by 5-reductase to the potent derivative, dihydrotestosterone, which induces virilization of the male external genitalia. In the female, aromatase mediates estradiol synthesis. In some species, aromatase expression is critical for ovarian development; its role in ovary development in mammals is less clear, although aromatase deficiency is associated with transdifferentiation of granulosa cells into Sertoli-like cells (72).
Sf1 is restricted to Leydig cells after 13.5 dpc and mediates expression of several genes encoding enzymes required for testosterone biosynthesis including StAR (steroidogenic acute regulatory protein), Cyp11a1, Cyp17 (cytochrome P450 hydroxylases), and 3?HSD (hydroxysteroid dehydrogenase). The factors required for Leydig cell determination and lineage development are incompletely understood. Moreover, their origin, whether from immigrant mesonephric cells or progenitors within the gonad, remains equivocal. Leydig cell fate appears to rely on paracrine signals. Dhh, a Sertoli-secreted factor, induces expression of Cyp11a1 (side-chain cleavage) in the Leydig cell (46). The Dhh signal is received by the receptor patched (Ptc1), which is expressed on Leydig cells. Dhh is thought to play a crucial role in Leydig cell differentiation and is an example of a paracrine signaling pathway identified between two developing cell types. Genetic analysis has placed Pdgfr- (platelet-derived growth factor receptor-) upstream of Dhh (73). Like Fgf9, Pdgfs induce mesonephric migration, and Pdgfr- is necessary in the gonad for full Cyp11a1 expression. The X-linked gene Arx (Aristaless-related homeobox) also influences Leydig cell development. However, it is expressed mainly in peritubular myoid cells, endothelial cells, and in the epithelium of the tunica albuginea (74).
Female Embryonic Gonadal Development
Relatively few genes have been shown to exhibit a female-specific pattern of gene expression early in gonadal development. To date, no gene has been shown to be a female-determining gene, as defined by conferring complete female-to-male sex reversal when mutated on an XX background, or by conferring male-to-female sex reversal after overexpression on an XY background. The Dax1 gene was initially suggested as a pro-ovarian, or anti-testis, candidate gene because its duplication on an XY background is associated with impaired testis development (75, 76). However, Dax1 loss of function on the XX background does not prevent ovary development (31). Subsequent studies have shown an unexpected role for Dax1 in testis development (77), indicating that its actions are highly dependent on the timing and level of expression.
Although male somatic tissue can survive in the absence of germ cells, the female gonad depends on migratory germ cells to populate the undifferentiated primordium (78). In the female gonad, germ cells begin entering meiosis at 13.5 dpc and initiate a dynamic paracrine relationship with supporting cells destined to become cumulus and granulosa cells (79).
A growing list of genes exhibit an ovary-specific expression pattern. For example, follistatin (Fst) is highly expressed in the developing ovary relative to the testis (19). Follistatin binds members of the activin/TGF?/bone morphogenetic protein (BMP) family. Thus, it may regulate the activity of one or more of these factors. Stra8 (stimulated by retinoic acid) is also selectively expressed in the developing ovary and is thought to play a role in regulation of meiosis (80). Targeted mutagenesis and transgenic overexpression experiments will clarify the functions of these factors.
There has been postulated a "Z factor" that could act to suppress pro-testis events carried out by autosomal or X-linked genes in both XY and XX backgrounds (81). The Z factor would act downstream of Sry in genetic males. Therefore, the Z factor would be repressed by Sry in the male, and independent of Sry on an XX genetic background. Loss of a Z factor would be sex-reversing on XX (female-to-male), but gain-of-function on XY may or may not exhibit sex reversal (male-to-female) depending on whether the Z factor can override the downstream events of Sry. One candidate for the Z factor is Wnt4 (Wingless-related integration site). Female Wnt4 null mice develop virilizing gonads and Wolffian derivatives (82). This gonadal phenotype reflects the action of Wnt4 to induce the migration of steroidogenic precursors of the adreno-gonad primordium into the anterior region of the gonad (83). Hence, misdirected adrenal steroidogenic cells become localized in the XX Wnt4 knockout gonad. Secondly, Wnt4 is down-regulated in males, whereas its expression remains strong in females where it prevents coelomic vascularization (a male feature, see Fig. 1B) through a molecular pathway that involves Fst and Bmp2 (19). By inhibiting vascularization, Wnt4 may prevent the proliferative growth expansion characteristic of male development. Thus, the homozygous knockout female gonad appears somewhat more male-like in shape and size. Externally, however, homozygous knockouts exhibit female genitalia. In humans, a loss-of-function mutation in WNT4 caused Mayer-Rokitansky-Kuster-Hauser syndrome, which is characterized by defective development of Müllerian derivatives (84). Duplication of a chromosomal region containing WNT4 was associated with a case of human XY sex reversal (85). However, overexpression of Wnt4 on an XY background does not lead to XY sex reversal in the mouse (83, 86). Thus, the major roles of Wnt4 in the female appear to include suppression of androgen-producing interstitial cells, inhibition of testis-like vascularization, and support of Müllerian derivatives. Wnt4 is also essential for kidney (87), mammary gland (88), and pituitary development (89), as well as normal functioning of the adrenal cortex (90).
A dominant insertional mutation led to identification of a female-to-male sex reversal phenotype in the Odsex (Ocular degeneration with sex reversal) mouse (91). The Odsex mouse was initially thought to lack a repressor for Sox9, allowing unabated Sox9 expression ectopically in genetic females. Therefore, one of the functions of a Z factor could be to suppress Sox9 action in the female gonad. However, it is also possible that regulatory elements contained in the inserted transgene promote activation of Sox9.
Nucleo-cytoplasmic shuttling of transcription factors may also provide an important regulatory step in sex determination. Organ culture experiments using the nuclear export blocker, leptomycin B, resulted in male-characteristic Sox9 and MIS expression in XX gonad organ cultures (92). Thus, growth factors may be able to modulate transcriptional control by altering the location as well as the activity of various transcription factors.
Central Questions and Future Directions
The molecular pathways directing cellular differentiation can be traced back to cells of pluripotential origin. Thus, development must occur through a series of commitment steps driven by cell-to-cell contacts and paracrine interactions. In some cases, committed cells initiate a cell-autonomous program, as perhaps illustrated by Sry and Sox9 actions in Sertoli cells. In other cases, differentiation is driven by secreted factors as illustrated by Dhh, Wnt4, and Fgf9. These cell autonomous and paracrine pathways are not mutually exclusive, and it is likely that most commitment steps integrate a series of internal and external signals. The sex determination pathway provides a unique paradigm for considering these events because the commitment steps lead to distinct cell types within a narrow time frame.
Despite impressive progress in this field, a number of challenging questions remain. What factors initiate Sry expression and what are its cellular targets? Are Sry and Sox9 expression linked directly or do they reflect parallel, partially redundant pathways? Given that Sox9 is sufficient to induce Sertoli cell development, what are its targets and how does it induce a network of genes they convey morphological changes characteristic of the testis? What are the genetic and molecular events that dictate ovary development? Although it is known that germ cells interact actively with somatic cells, what are the molecular and cellular events that control germ cell replication and entry into meiosis? How do meiotic germ cells foster ovary development? Implicit in these questions is the critical feature of timing during gonadal development. Once the Sertoli program is initiated, the developing testis induces mitotic arrest of germ cells. On the other hand, if Sertoli development is delayed, as occurs with various Sry alleles and mouse genetic backgrounds, germ cells begin to enter meiosis, and this step may further impair testis development, leading to mixed testis/ovary tissue or gonadal dysgenesis. Thus, future studies need to carefully assess pathways that control the timing and level of expression of regulatory factors.
Further understanding of these molecular and cellular functions in the gonad will provide examples of paradigms used to control cellular differentiation in other tissues. This knowledge will also be useful for the characterization of intersex cases and perhaps additional causes of idiopathic infertility. Parallel investigation of human mutations and animal models can accelerate progress in this field by identifying candidate genes and clarifying their roles in development.
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