The chromosomal sex of the embryo is established at fertilization. However, 6 weeks elapse in humans before the first signs of sex differentiation are noticed. Sex differentiation involves a series of events whereby the sexually indifferent gonads and genitalia progressively acquire male or female characteristics. Believed initially to be governed entirely by the presence or absence of the SRY gene on the Y chromosome, gonadal determination has proven to rely on a complex network of genes, whose balanced expression levels either activate the testis pathway and simultaneously repress the ovarian pathway or vice versa. The presence or absence of primordial germ cells, of extragonadal origin, also has a sexually dimorphic relevance. Subsequently, internal and external genitalia will follow the male pathway in the presence of androgens and anti-Müllerian hormone (AMH), or the female pathway in their absence. Here we review the sexually undifferentiated stage of embryonic development, and the anatomic, histologic, physiologic and molecular aspects of the fetal sexual differentiation of the gonads, the internal reproductive tract and the external genitalia. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.
Genital sex differentiation involves a series of events whereby the sexually indifferent embryo progressively acquires male or female characteristics in the gonads, genital tract and external genitalia. Sex development consists of several sequential stages. Genetic sex, as determined by the chromosome constitution, drives the primitive gonad to differentiate into a testis or an ovary. Subsequently, internal and external genitalia will follow the male pathway in the presence of specific testicular hormones, or the female pathway in their absence. Since the presence of the fetal testis plays a determining role in the differentiation of the reproductive tract, the term "sex determination" has been coined to designate the differentiation of the gonad during early fetal development.
The urogenital ridges are the common precursors of the urinary and genital systems and of the adrenal cortex (1). In the human, they develop during the 4th week post-fertilization at the ventral surface of the cranial mesonephroi, and are formed by intermediate mesoderm covered by coelomic epithelium. Each urogenital ridge divides into a urinary and an adreno-gonadal ridge in the 5th week (Table 1). The adreno-gonadal ridge is the common precursor of the gonads and adrenal cortex. The gonadal ridge is bipotential and can develop into an ovary or a testis. Gonads are subsequently colonized by the primordial germ cells, of extra-gonadal origin. The mesonephroi also give rise to components of the internal reproductive tract and of the urinary system.
Several general transcription factors belonging to the large homeobox gene family play an important role in the stabilization of the intermediate mesoderm and the formation of the urogenital ridges (Table 2). Mice in which Lhx1 (4), Emx2 (5, 6) or Pax2 (7) have been inactivated fail to develop urogenital derivatives. Most of these ubiquitous factors are essential for the development of other vital embryonic structures. However, another LIM homeobox gene, Lhx9, seems to be essential only for the proliferation of somatic cells of the gonadal ridge (8) by interacting with Wt1 to regulate Sf1 (9). LHX9 expression increases in both XX and XY undifferentiated gonads, and then decreases as Sertoli and granulosa cells differentiate (10, 11). Several other factors are involved in cell proliferation in the gonadal primordium both in XX and XY embryos. For instance, impairment of the signaling pathway of the insulin/insulin-like growth factor family in mouse knockout models with disrupted Insr, Igf1r and Insrr leads to a significant reduction of the size of adreno-gonadal ridges in both XX and XY embryos (12). Also in mice with a knockout of Tcf21, gonads are severely hypoplastic in both XX and XY fetuses (13). GATA4 (14) and the homeoproteins SIX1 and SIX4 are also essential for early proliferation of gonadal precursor cells and for FOG2- and SF1-regulated SRY expression (15). The Notch signaling pathway is also involved in somatic cell lineage commitment during early gonadogenesis in mice. Conditional knockout of Numb and Numbl (antagonists of Notch signaling) in the undifferentiated gonad results in disruption of the coelomic epithelium and reduction of somatic cell numbers in the gonads (16). Finally, NRG1 is also required in a dose-dependent manner in order to induce somatic cell proliferation in the gonads (17). Since cell proliferation is more important in the male than in the female early developing gonad (18, 19), sex-reversal is often observed in XY embryos with an alteration of gonadal cell proliferation (12). It has been suggested that this is due to a reduction in the number of SRY-expressing pre-Sertoli cells, resulting in very low levels of SRY expression that are insufficient to trigger testicular differentiation (discussed in ref. (20).
The differentiation of the gonadal ridge from the intermediate mesoderm requires the expression of sufficient levels of WT1 and SF1. WT1 was initially isolated from patients with Wilms' tumor, an embryonic kidney tumor arising from the metanephric blastema. By alternative splicing and alternative translation initiation, WT1 encodes more than 20 isoforms of a zinc-finger protein acting as transcriptional and/or post-transcriptional regulator (20). The -KTS splicing variant of WT1, lacking the three amino acids lysine (K), threonine (T) and serine (S) at the end of the third zinc finger, is required for cell survival and proliferation in the indifferent gonad, whereas the +KTS variant is involved in the regulation of SRY expression (21). The first indication of a role for WT1 in gonadal and renal development was its expression pattern in the urogenital ridges (22). During gonadal differentiation, WT1 is expressed in the coelomic epithelium and later in Sertoli and granulosa cells (23). In mice with a knockout of WT1, neither the kidneys nor the gonads develop (24). In humans, mutations in the WT1 gene do not completely prevent urogenital ridge development but may result in gonadal dysgenesis associated with nephroblastoma (Wilms' tumor) and/or nephrotic syndrome owing to glomerular diffuse mesangial sclerosis (25-27).
SF1, also known as Ad4BP or FTZF1 (HGNC approved gene symbol: NR5A1), initially described as a regulator of steroid hydroxylases, is an orphan nuclear receptor expressed in the hypothalamus, the pituitary, the gonads and the adrenal glands (reviewed in refs. (28-30). In mice with a knockout of the SF1 gene, the intermediate mesoderm is not stabilized and the gonadal and adrenal primordia soon degenerate (31). SF1 also plays an important role in spermatogenesis, Leydig cell function, ovarian follicle development and ovulation, as demonstrated by a gonad-specific disruption of SF1 (32). A recurrent heterozygous p.Arg92Trp variant of the gene is associated with testicular development in XX subjects (33, 34). WT1, through interaction with CITED2 (35, 36), and LHX9 (8) regulate the expression of SF1 upstream of the gonadal development cascade. GATA4 and SOX-family factors also regulate SF1 expression in the gonad (28). In humans, the phenotype resulting from SF1 mutations does not exactly match that of Sf1 knockout mice: the clinical spectrum includes severe and partial forms of testicular dysgenesis, anorchidism, and even male infertility in normally virilized individuals; adrenal insufficiency is not always present. In 46,XX females, SF1 mutations have been described in patients with primary ovarian insufficiency (29, 30). SF1 is one of the increasing number of examples of dosage-sensitive mechanisms in human sex differentiation, since mutations at the heterozygous state are sufficient to induce sex reversal in XY individuals (reviewed in refs. (29, 30).
Recent studies using single-cell RNA sequencing (scRNA-seq) has shed light on the initial steps of lineage trajectories and cell fate in the developing gonads (1, 37). A subset of cells of the coelomic epithelium expressing GATA4, SF1 and WT1 are likely to be the precursors of the somatic lineages of the undifferentiated gonads: both the supporting (Sertoli and granulosa) and the steroidogenic (Leydig and theca) cell populations of the differentiating gonads seem to derive from SF1 and WT1-expressing cells present in the genital ridge (1, 37, 38).
In the 4thweek, PGCs have migrated and are present in the yolk sac near the base of the allantois. They can be identified by their expression of alkaline phosphatase, OCT3/4 and the tyrosine kinase receptor C-KIT (Fig. 1A) (40). Subsequently, PGCs become embedded in the wall of the hind gut, gain motility and migrate through the dorsal mesentery to reach the gonadal ridges in the 5thweek (Fig. 1B). Early migration of PGCs is dependent on the expression of interferon-induced transmembrane proteins 1 and 3 (IFITM1 and IFITM3) in the surrounding mesoderm (51). During migration, PGCs proliferate actively but do not differentiate (40). Germ cell migration through the dorsal mesentery to the gonadal ridges and survival/proliferation in both XX and XY embryos is driven by signaling between kit ligand (KITL, also known as Stem cell factor [SCF], Steel factor or mast cell growth factor [MGF]), which is expressed in somatic cells of the gonadal ridge and the hind gut along the pathway of PGC migration, and its receptor present in germ cells, C-KIT (Fig. 1) (52). PGC migration and genital ridge colonization is also dependent on stromal cell-derived factor 1 (SDF1, also known as CXCL12) and its receptor CXCR4 (53) and on interactions with extracellular matrix proteins, like fibronectin and laminin, while proliferation and/or survival involve many other factors (39, 40, 52, 54). 2b1af7f3a8