Perspective: Reproductive Tract Development—New Discoveries and Future Directions

At first glance, sexual reproduction among animals with sex organs seems to be accomplished by a wide variety of species-specific processes. Fertilization can be internal or external requiring no intromission, and animals can be oviparous, viviparous, and even ovoviviparous. The number of viable offspring per mating can vary from one to thousands, and gestation can take anywhere from days to nearly 2 yr to complete, depending upon the species. Ovulation itself can be reflex in nature, induced by the act of copulation, or it can be completely unrelated to the mating process. Closer examination of the process, however, reveals many unmistakable similarities in this array of phenotypic differences. Mature sperm and oocytes have to be produced by testes and ovaries and brought into close proximity with one another. If fertilization results, there needs to be a suitable place for embryonic and fetal development to take place, and there must be a process whereby the new member of the species is introduced into its environment. Clearly, there are species-dependent solutions to these problems, but the overarching theme is common among all sexually reproducing animals: namely, all species need a mechanism to ensure that males and females are produced in nearly equal numbers in each breeding cycle to ensure future propagation of their kind. Subsequent sex-dependent gene expression functions to develop two distinct male and female phenotypes. The development of two distinct reproductive systems is, in fact, accomplished by basic molecular mechanisms that share a great deal of homology across species. Studies conducted in one species, therefore, can yield extremely relevant information to others, just as is true for the remarkable conservation of cell cycle control proteins, transcription machinery, and DNA repair enzymes from yeast to humans. These processes are initially completely independent of the sex of the developing animal, and they produce sexually indifferent structures with the capacity to be either the male or female reproductive tracts. That is, both male and female embryos develop Wolffian ducts and Müllerian ducts, but only one of these precursor ductal systems will survive as a functional reproductive tract. At a specific point in embryonic development, the genetic sex of the animal is declared, the organization of testes or ovaries is initiated, and the subsequent differentiation of male and female reproductive tract phenotypes is begun. What follows is an overview of much of what is known about the process of reproductive tract development and a discussion of some interesting challenges for the future. The development of the reproductive tract is a process that begins shortly after the creation of a zygote at fertilization and continues throughout incubation in the case of oviparous species and gestation for viviparous animals. The duration of these periods varies among species. For example, in the human, the embryonic period spans weeks 2–8 of gestation, whereas the remaining 32 weeks are the fetal period; but in rodents, the embryonic and fetal periods are of near equal length and comprise a total of 3 weeks. However, the process is not complete until after birth as adolescents mature into adults with the capacity to reproduce sexually. This Perspective will focus on the development of embryonic and fetal reproductive tracts. The development of the adult reproductive tract is the subject of other Perspectives in this issue of Endocrinology. The reproductive tract is defined as the structures derived from the Müllerian ducts in females, those from the Wolffian ducts in males, the testes and ovaries, and the relevant external genitalia arising from the urogenital tubercle and labioscrotal structures. The ducts and gonads have their embryonic origins in the tissues of the urogenital ridge that arise from the enlarging intermediate mesoderm that forms upon gastrulation, becomes populated with germ cells, and undergoes continued growth and morphogenesis during the embryonic period (Fig. 1A). A, Formation of the urogenital ridge in the embryo. The development of the intermediate mesoderm into the urogenital ridge during the embryonic period sets the stage for later differentiation into the gonads, Wolffian and Müllerian ducts. Under the control of several sex-independent genes, the ridge takes shape awaiting the sex-dependent signals that will evoke gonadal identity and the subsequent reproductive duct growth. B, Gonadogenesis and reproductive duct development. Later in embryonic development at least six gonad-determining genes (SF-1, Lim 1, WT-1, GATA-4, Lhx 9, and Emx2) direct formation of the indifferent gonads from the intermediate mesoderm. Absence of any of these genes blocks gonadal development. Thereafter, in males the genes necessary for normal testes, Sry, SF-1, Sox-9, and Dhh act to produce the gonad. The secretion of testosterone and MIS ensure the development of the normal male phenotype. In females, on the other hand, the lack of male determining genes allows for the development of ovaries. Wnt-4, Fa, and several of the Hoxa genes promote the proper growth and development of the Fallopian tubes, uterus, cervix, and vagina. A number of genes encoding transcriptional regulators and secreted ligands have been identified as critical for the development of embryonic intermediate mesoderm. The homeobox containing gene transcription factors Lim1 (1), Lhx 9 (2), and Emx2 (3) are expressed beginning at gastrulation in visceral and lateral folds that make up the intermediate mesenchyme. Disruption of the Lim1 gene produces a disorganized structure with reduced expression of Pax-2, a paired box transcription factor, which becomes a marker of the Wolffian duct (4), and the homeobox gene Hox 6b (5). Hox-6, and a number of other Hox genes, are vertebrate homologs of Drosophila melanogaster homeotic genes that define the developing body axes. Selective disruption of Hox genes leading to the loss of tissues in the embryo has identified structures dependent upon their function. Many, if not all, of these genes are also involved in the elaboration of the urinary tract tissues and gene deletions lead to renal agenesis as well as reproductive tract anomalies. It is not uncommon for patients with congenital anomalies of the reproductive tract to have associated urinary tract defects, implying a common molecular pathophysiology occurring early in embryonic life. The expression of Hox transcription factors occurs along the length of the developing Müllerian duct and it continues in the Fallopian tubes (Hoxa 9) uterus (Hoxa10, 11), cervix (Hoxa11) and upper vagina (Hoxa13) in adults (6). Although all of these tissues are Müllerian-derived structures, these genes are not unique to the reproductive tract of either sex. Another gene whose expression is critical for normal reproductive tract determination is Wnt-4. This ligand is expressed in the coelomic epithelium that invaginates to form the Müllerian duct and then in the mesenchyme surrounding the duct. Wnt-4 is a member of the Wg/wnt family of secreted proteins that function in intracellular communication via the frizzled family of receptors and are required for early pattern formation, cell fates, and polarity (7, 8). Wnt4 knockout has no consequence for the male reproductive tract because it is not expressed in Wolffian ducts, but females in whom the gene was deleted do not have Müllerian duct-derived internal reproductive structures. Although Wnt-4 is required for Müllerian duct formation in both males and females, and thus, is not sex specific, it is only in females that its functional expression persists. Nephrogenesis (8, 9) depends upon Wnt-4 because both males and females die at birth with renal agenesis. The association of Müllerian and renal agenesis in Rokitansky Hauser syndrome in which the uterus is absent and the kidney on one side is absent, abnormal, and/or ectopically positioned in the pelvis, implicates Wnt-4 or its pathway in this disorder. Bilaterality is lethal, however, and patients who normally have unilateral defects are probably reflective of a hypomorphic mutation. Wnt-7a, another member of the family, is also required for Müllerian duct differentiation. Viable mice lacking Wnt-7a ligand have limb defects and are infertile; males had persistent Müllerian ducts attributed to the failure of Müllerian Inhibiting Substance (MIS) type II receptor expression and females had poorly differentiated Müllerian duct derived structures (10). Wnt-7a, therefore, normally mediates the expression of the MIS type II receptor (see below) allowing complete regression of the Müllerian duct in males. Poor Müllerian duct development in females implies a role for Wnt-7a in the subsequent differentiation of this tissue. It is interesting to note that diethylstilbestrol, a synthetic estrogen known to cause uterine anomalies in fetal females (11), suppresses Wnt-7a expression (12) and alters the expression of Hoxa 9 and 10 (13). Although the exact mechanism involved with DES is unknown, these findings may, in part, explain the molecular pathophysiology of the defects observed in the so-called DES babies (14). The effects of disrupting Wnt-7a on the female tract are not due to any affect of homozygous deletion of the gene on gonadal function or hormonal regulation since the ovaries undergo normal follicular growth, ovulation, and cycling, but to a defect intrinsic to the Müllerian duct. At the end of this embryonic phase the anlagen of all major structures are present but organogenesis is incomplete and genetic males and females are virtually indistinguishable. The genes thus far identified are involved in processes intrinsic to tissue patterning and axis formation rather than the sex-specific development, which occurs after gonadal differentiation. The identification of these early genes and the nature of the genes regulated by them open new areas of investigation and link the development of the urinary and genital tracts at this stage of their ontogeny. The Wilms’ tumor factor, WT1 (15), and the steroidogenic factor, SF-1 (16), are other examples of factors that when mutated fail to develop both urinary tracts and gonads with subsequent consequences for reproductive tract development. Both will be important in regulating genes such as MIS that are important in sex differentiation later in development, as well, as is GATA-4, another transcriptional regulator, also expressed in primitive gonads in both male and female embryos (17). In males, GATA-4 expression continues in somatic cells of the testes throughout development but is transient in the ovary suggesting a role in sexually dimorphic development in the gonads (17). Reproductive tract development becomes sexually dimorphic, producing two distinct phenotypes emerging under the control in mammals, for example, of two distinct pairs of sex chromosomes, XX for females and XY for males. This process, which is well cataloged anatomically in nearly all species, begins in the embryo and continues well after birth and can take years to complete, as is the case in humans. A number of sex-specific genes have been found to regulate gonadal differentiation and subsequent male and or female reproductive duct development. It is at the transition from embryonic life, which is completely independent of the sex chromosomes, to fetal life that the genetic sex of embryos is declared as primordial gonads are induced to differentiate into testes or ovaries according to their chromosomal complement to direct further development (Fig. 1B). In males, the newly formed testes produce testosterone, which stimulates the differentiation of the Wolffian ducts into the epididymides, vas deferens, and the seminal vesicles while MIS, also known as anti-Müllerian hormone, is secreted by the fetal Sertoli cells and ablates the Müllerian duct. In females, in the absence of MIS, the Müllerian ducts, which have formed from an invagination of the coelomic epithelium, become the uterus, cervix, upper third of the vagina, and the Fallopian tubes. The coelomic epithelium persists as the lining of the ovaries. Because testosterone is required for the development of the Wolffian ducts, in its absence they atrophy. There does not appear to be a female homolog of MIS for the Wolffian ducts. The external genitalia of both sexes arise from the genital tubercle and the urogenital folds that differentiate from the underlying mesenchyme in the embryonic period. In males dihydrotestosterone, a steroid 5-α reduced metabolite of testosterone, stimulates growth of the phallus. There are two 5-α reductase enzymes, called 1 and 2, that convert testosterone to DHT (18). Mutations in type 2, which is expressed in liver and gonad and genital skin, results in male pseudo-hermaphroditism (19). The female external genitalia develop autonomously in the fetus requiring no hormonal stimuli at this stage. The testis determining factor gene product, SRY (sex-related gene on the Y chromosome), which is expressed in the intermediate mesoderm at the end of embryonic development, is a nuclear transcription factor that is absolutely required to produce normal testes in mammals, as shown in Sry mutated mice (20), and mutations in the human homolog of this are associated with sex reversal (21). SRY is assumed to be a transcription factor because its homology to the HMG family but a direct target for the transcriptional regulation has yet to be described (22). Structural mutations and transcriptional analyses point to a requirement for DNA binding and bending for functional activation by SRY (22, 23). SRY may, more importantly, provide a nuclear scaffold to allow better access of transcription factors to the promoter regions of genes required for male-specific differentiation. In mice Desert hedgehog, Dhh, is another male specific gonadal gene, which functions after the action of Sry to signify early testicular differentiation. Dhh regulates Sertoli-germ cell interactions; homozygous knockout males have testes but they are azoospermic (24). Although not required for testis formation, estradiol does play a role in normal spermatogenesis. Adult male mice in which the estradiol α-receptor has been mutated have greatly reduced sperm counts and are infertile (25). To date, no such gene has been identified for the generation of ovaries from indifferent gonads. DAX-1 (deleted in adrenal hypoplasia congenita from the X chromosome (26–28) was originally thought to be related to ovarian determination because a duplication of the dosage-sensitive sex reversal (DSS) locus on the X chromosome, which also contains the DAX-1 site, caused male to female sex reversal (29, 30). However, female mice deficient in DAX-1 have normal phenotypes and are fertile, but males exhibit abnormal testicular development and ultimately infertility (26). Therefore, DAX-1 is necessary for the maintenance of normal seminiferous tubule function in the adult (26). DAX-1, is a member of the nuclear hormone receptor superfamily and a potent transcription repressor. Recent studies show Dax-1 functions to suppress testicular development by blocking the transcriptional activity of SRY (31) indicating that normal gonadal development, particularly in the male relies on a proper balance of Sry and Dax-1 gene activities. DAX-1 also suppresses SF-1 (32) and GATA-4 function by engaging the corepressor NCoR (33). Wnt-4, previously described for its role in Müllerian duct determination in males and females, is also expressed in indifferent gonads. However, Wnt-4 expression ceases in the testes but it is preserved in the ovaries where it maintains oocyte number (8). The fact that Sox 9 (SRY-related homeobox protein 9) is mutated with patients with sex reversal associated with campomelic dysplasia (34), provides further evidence of its important role in male reproductive tract development (Fig. 1B). Once the testis begins to differentiate after the action of Sry, the fetal Leydig cells secrete testosterone and Sertoli cells release MIS. These two hormones have widely differing functions and mechanisms of action, but it is their combined that produce a normal male internal reproductive tract. to its intracellular receptor to function as a transcriptional leading to the and differentiation of the Wolffian duct into the vas deferens, and seminal testosterone or its functional receptor these do not These receptors are present first in the mesenchyme of the developing duct and the action of these cells, by mechanisms in to the the cell to its proper and have in both Wolffian and Müllerian ducts, the role by the mesenchyme in the differentiation of these ducts Although the genes regulated by testosterone in the Wolffian duct to be the of male tract development on this steroid hormone is of nature, testicular this In the genetic males with testosterone receptors mature as phenotypic have testes, they lack Müllerian structures because of the action of MIS (see but they also lack any Wolffian duct The other well fetal testicular of to the reproductive tract is MIS. This protein is among species and it is regulated in a and in and adult Sertoli cells, and and adult This hormone, whose was by the of is a member of the family of growth in embryonic the of what called the Müllerian or the embryonic testicular in female embryos sexual differentiation had found that the animals they of Wolffian ducts and regression of the Müllerian ducts. the female embryos but not cause regression of the Müllerian ducts leading to that there must be a testicular hormone in to testosterone that was for Müllerian duct The MIS ligand with the MIS type II receptor and receptors that are expressed on the cell of the fetal Müllerian duct and by Sertoli cells and cells of both embryonic and adult gonads. with mutated MIS type II receptor have Müllerian ducts, as do patients with persistent Müllerian duct syndrome the mutations in MIS type II receptor gene found in patients with this a deletion in 10 was the common Because the homozygous female MIS type II receptor knockout mice have normal it appear that MIS is not required for either or fetal and embryonic development. to the type receptor that or be in this with the more evidence for function in the Müllerian duct mesenchyme to because male mice deleted for do not have Müllerian ducts The of MIS expression that occurs in the fetal male is by a of transcription factors in which SF-1 and WT1 with GATA-4 and Sox 9 In disruption of the gene, but not one of two SF-1 binding in the MIS promoter reduced MIS expression in and caused sex reversal indicating that a SF-1 is required for MIS expression In fetal females and after in males, the Dax-1 suppresses the SF-1 and GATA-4 activation of the MIS gene (33). A fetal in reproductive tract development another sexually dimorphic phenotypic the of the gonads. The ovaries in the in place by the to the while the testes into the as the For proper of the ovaries, therefore, the in the absence of testosterone and the In males, the is by the action of testosterone, which also stimulates the to complete the into the It is that at least two both of these Leydig cell are required for testis and regression of the in males, testosterone and is an growth factor which with a factor receptor These two growth, a process necessary for testicular and also be involved in this process because of these genes to testes and abnormal In male knockout the to develop therefore, the testes in the If the receptor is also deleted in the the does not under the action of testosterone and the testes a near the a normally by the ovaries of the gene due to to can lead to suggesting that a proper balance of and estrogen in must be for normal male phenotypic development. In the absence of testes and testosterone and MIS, the Müllerian ducts develop into the Fallopian tubes, uterus cervix, and the upper third of the vagina. The coelomic epithelium, which invaginates to form the Müllerian also the the expression of both of estradiol receptor in the mesenchyme and the epithelium of the Müllerian are not required for Müllerian duct development in a animals receptor for both the and forms of the estradiol receptor show on the development of any tissue in a male or female tract development normal after birth the uterus, Fallopian tubes, cervix, and vagina lack normal to estradiol and are that fetal estradiol another ovarian is required for Müllerian duct development is found in the SF-1 knockout animals. The females lack and ovaries but have normal reproductive tracts In the males also lack gonads and but are sex The absence of testosterone therefore, to of the Wolffian duct and Müllerian duct development thus the of the female as an pathway of differentiation and development. the genes known to be required for normal Müllerian duct development, are sex Wnt-7a, for example, the transcription of the type II receptor for MIS and males lacking functional Wnt-7a have Müllerian ducts, whereas females are infertile because they have later poorly differentiated Fallopian tubes and The and of related to and to the organogenesis and differentiated function of tissues the are and will be as the of the human and other become The of a well to genetic genes, proteins, and either or by with to has and the of and the of to human and It is no necessary to a new gene but to genes in a In this new as an of have but not all, of the embryonic and fetal genes and factors involved in normal sexually dimorphic reproductive tract development. of the genes either transcription factors or secreted but must which genes the transcription factors regulate and the molecular consequences of of the that it will be to a number of of a more For example, does testosterone direct the Wolffian development for What early growth and differentiation of the female reproductive tract that no sex required at all for reproductive tract development birth the of receptors in Müllerian there be a sex-specific role for estradiol in the embryo because both male and female are in estrogen from the and receptors are present in the mesenchyme of the reproductive is to the signals from the mesenchyme to the epithelium to or suppress growth and differentiation. and to gene expression and to of secreted proteins will these to be In that and analyses will further and will be to new these new at it is not to the to the that have reproductive for These in will open to other had not