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Abstract

Wolffian ducts (WDs) are the embryonic structures that form the male internal genitalia. These ducts develop in both the male and female embryo. However, in the female they subsequently regress, whereas in the male they are stabilised by testosterone. The WDs then develop into separate but contiguous organs, the epididymis, vas deferens and seminal vesicles. Recently, considerable progress has been made in identifying genes that are involved in these different stages of development which is described in this review. In addition, WD development in (atypical forms of) cystic fibrosis and intersex disorders, such as the complete androgen insensitivity syndrome, 17β-hydroxysteroid dehydrogenase deficiency and LH-receptor defects, is discussed. The apparent increase in male reproductive tract disorders is briefly discussed from the perspective of the potential endocrine-disrupting effects of the numerous chemicals in the environment to which the developing male foetus can be exposed.

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Introduction

Wolffian ducts (WDs) are the embryonic structures that form the male internal genitalia. Their development is therefore essential to male fertility. In addition, the WD plays a crucial role in kidney development, both in the male and female embryo, as well as in development of the Müllerian duct (MD). The MD is the precursor of the female reproductive tract which has been subject of a previous review <1>. In recent years, considerable progress has been made in elucidating the genes involved in regulation of WD development, as summarised in the present review. Also discussed is the pathophysiology of WD development in cystic fibrosis and intersex disorders, together with reference to the potential effects of endocrine disrupters on WD development. Medline was searched using the terms ‘Wolffian duct’, ‘epididymis’ and ‘vas deferens’ and the MESH term ‘mesonephric duct’ to identify articles on these subjects; references from articles found in the original search were also used.

Embryology of the Mesonephric Tubules and Wolffian Ducts

Two pairs of unipotential ducts, the WDs (also known as mesonephric ducts) and MDs (also known as paramesonephric ducts) are anlagen of the male and female reproductive tracts respectively, and form independent of the genetic sex of the embryo. The WD forms from the intermediate mesoderm at E9 in mice, in craniocaudal succession. It initially forms as an extension of the pronephric duct in the region of the future forelimb buds, and grows caudally to the cloaca. The WD induces the formation of mesonephric tubules in the mesonephric mesenchyme, which extend to the epithelial cells of the gonad in both males and females <2> (fig. 1a). In rodents, the four to six most cranial tubules bud from the WD, whereas the more caudal tubules are close to, but not connected to the WD <3>. The caudal tubules degenerate in males and females, but the cranial tubules persist in males to form the efferent ducts <3>. The ureteric bud branches from the WD posteriorly, to form the kidney through interaction with the metanephric mesenchyme.


Schematic drawing of the development of the male genital tract in humans. a Schematic drawing showing the excretory system of the gonad and MES at 8 weeks gestation. SEM anastomose to form the RT, which is connected to the MT that drain into the WD. Mesonephric tubules that are not connected to the testis degenerate. b Male urogenital tract in a newborn. The mesonephric tubules and WDs have developed into ED, EPID, VAS and SV. BL = Bladder; ED = efferent ducts; EPID = epididymis; MD = Müllerian duct; MES = mesonephros; MT = mesonephric tubules; RT = rete testis; SEM = seminiferous cords; SV = seminal vesicle; UR = urethra; VAS = vas deferens; WD = Wolffian duct.


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The WD differentiates between weeks 9 and 13 of gestation in the human male embryo. The proximal part coils and forms the epididymis, whereas the distal part forms the vas deferens (figs. 1b, 2). The seminal vesicles develop from lateral outgrowths of the caudal end of the vas deferens. In females, the mesonephric tubules and WDs degenerate, although remnants may be present in the form of an appendix vesiculosa, epoophoron, paroophoron or duct of Gartner.


Development of the foetal rat WD. Macroscopic photos of the WD at E18.5 (a), E19.5 (b) and E20.5 (c) showing growth and coiling of the duct. Scale bars = 1 mm.


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The MD forms in an anterior to posterior manner by invagination of the coelomic epithelium of the mesonephros, between E11.5 and E12.5 in mice <2>, and between 6 and 7 weeks of gestation in humans <4>. The presence of the WD appears to be essential to induction of MD formation. When caudal outgrowth of the WD is blocked before it has reached the area of coelomic epithelium from which the MD normally forms, the MD fails to develop <5>.

Genes Involved in Early Formation of the Wolffian Ducts

The use of mouse knockout models has provided much insight into genes involved in the development of WD. However, it has to be kept in mind that for many of these genes the relevance to WD development in humans still needs to be investigated.

Several genes are involved in the initial development of the WD and MD, which is similar in males and females (fig. 3). Pax2, a transcriptional regulator of the paired-box family is expressed in the epithelium of the mesonephric tubules as well as the WD and MD. Pax2-deficient mice lack kidneys and genital ducts, but the gonads develop normally <6>. The anterior portion of the genital ducts forms but subsequently degenerates, whereas mesonephric tubules never develop <6>. Pax8 is coexpressed with Pax2 and has redundant functions in urogenital development <7>. Disruption of Pax8 does not result in urogenital abnormalities, but in mice lacking both Pax2 and Pax8, the WD fails to develop, and cells that normally express Pax2 undergo apoptosis <7>. These findings suggest that Pax2 and Pax8 proteins are required for mesenchymal–epithelial conversion, a process which is essential for the formation of WD and mesonephric tubules <6, 7>.


Genes involved in WD development. Overview of genes implicated in early formation of the WD (occurring in both sexes), androgen-dependent stabilisation of the WD and differentiation of the WD into distinct subsections. RAR = retinoic acid receptor.


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Lim1, which encodes a homeodomain transcription factor, is also expressed in epithelium of the mesonephric tubules, WD and MD <8>. Mice that lack this gene, do not have WD and MD derivatives. Lim1 may play a role in the formation or very early differentiation of MD and WD epithelium <8> and may act as a co-factor co-operating with Pax2 and Pax8<7>. Emx2 is also required for the formation of both pairs of genital ducts. It is expressed in the epithelium of the WD at E9.5, epithelium of the mesonephric tubules at E10.5, and the epithelium of the MD at E12.5 <9>. In Emx2 null mice, the WD and mesonephric tubules appear normal at E10.5, but the WD has started to degenerate by E11.5 and no MDs are present at E13 <9>. Retinoic acid signalling also plays a role in WD and MD development. Compound null mutations of retinoic acid receptors αand γ, or subtypes of these receptors, are associated with agenesis or dysplasia of the epididymis, vas deferens and seminal vesicles <10>. Mutations of these and other (sub)types of retinoic acid receptors also result in abnormalities of the MD <10>.

Results from studies of Wt1 null mice show that Wt1 is required for the formation of caudal mesonephric tubules, but not the cranial tubules that later form the efferent ducts <3>. The caudal tubules are thought to derive from the mesonephric mesenchyme, but the cranial tubules may (partly) derive from the WD and development of these tubules may be androgen-dependent <3>.

Differentiation of Sections of the Wolffian Duct

Region specific expression of several homeobox genes is important for differentiation of the WD into morphologically and functionally distinct structures: the epididymis, vas deferens and seminal vesicle. The Drosophila Abdominal B-related homeobox genes appear to be involved in defining tissue boundaries between these structures in mice. In the male mouse,Hoxa9andHoxd9are expressed in the epididymis and vas deferens,Hoxa10 and Hoxd10 mainly in the caudal epididymis and throughout the vas deferens, Hoxa11 in the vas deferens, and Hoxa13 and Hoxd13 in the caudal portion of the WD and seminal vesicles <11,12,13,14,15>. Studies on cultures of the urogenital sinus suggest Hoxa10 expression is not regulated by androgens <13>.

Mutations in Hoxa10 result in anterior homeotic transformation of the WD <16>; the distal epididymis and proximal vas deferens show morphological characteristics of more anterior segments. Disruption of Hoxa11 also results in a homeotic transformation of the vas deferens towards an epididymis-like phenotype <11>. Disruption of Hoxd13 alone leads to reduced size and reduced clefting of the seminal vesicles, whereas Hoxa13+/–/Hoxd13–/– mice display a more severe phenotype, with severely hypoplastic seminal vesicles <15>.

Genes Involved in Androgen-Dependent Development of the Wolffian Duct

Androgens and the Androgen Receptor

The WD once formed, will regress unless it is actively stimulated to grow. Testosterone is thought to be the sole factor responsible for stabilisation of the WD <17>. Leydig cells start producing testosterone at 8 weeks of gestation in humans, and at E15.5–E16.5 in rats <18>. Testosterone is thought to be secreted directly into and down the WD by diffusion <19>. Jost <17> demonstrated that castration of male rabbit foetuses before sexual differentiation has begun, results in regression of the WDs. However, a crystal of synthetic androgen placed in the abdominal cavity of castrated foetuses, whether male or female, prevents WD regression. Unilateral castration only results in atrophy of the WD on the operated side, suggesting the effect of a high local concentration of androgen. Testosterone interacts with the androgen receptor (AR) to exert its effects. Mice lacking AR show agenesis of the epididymis, vas deferens and seminal vesicles <20>.

During WD development, AR expression itself is regulated by androgens <21>. The male pattern of AR expression is seen in the WD of female rat foetuses exposed to dihydrotestosterone (which results in WD stabilisation), and at E21 the AR is not expressed in the WD mesenchyme of male foetuses exposed to the anti-androgen flutamide <21>. In the normal male rat foetus, AR is first expressed in the mesenchymal cells and subsequently in the epithelial cells <21>. This induction progresses in a proximal to distal manner.

Epithelial–Mesenchymal Interactions

The fact that the AR appears in the mesenchyme before it is expressed in the epithelium, suggests that androgens initially induce their effects on the epithelium via signals from the mesenchyme. This idea is supported by studies showing that, in tissue recombinants, seminal vesicle mesenchyme can induce proliferation and differentiation of AR negative epithelium from androgen insensitive tfm mice <22>. However, epithelial AR expression is a prerequisite for some functions, such as the synthesis of secretory proteins <22>. Epithelial AR expression is thought to be induced by the mesenchyme <23>.

Interactions between epithelium and mesenchyme are essential for the development of many organs, including the WD. The mesenchyme determines the fate of the epithelium. Experiments using tissue recombinants have shown that epithelium from both the ureter and the upper WD, which normally develops into the epididymis, can be redirected to develop morphological and functional characteristics of seminal vesicle epithelium when recombined with seminal vesicle mesenchyme <24, 25>. Conversely, growth of the mesenchyme is dependent on signals from the epithelium, as was demonstrated by failure of grafts of seminal vesicle mesenchyme alone to develop <24, 25>. The AR presumably plays a role in the regulation of factors that mediate or affect epithelial–mesenchymal interactions. Growth factors appear to be examples of such mediators.

Growth Factors

Epidermal growth factor (Egf) expression in the mouse WD appears to be regulated by androgens <26>. Egf mRNA levels increase during WD development in the male foetus and are higher in the male than in the female reproductive tract at E18. The levels can be increased in the female reproductive tract by treatment with testosterone (which also results in WD stabilisation) and reduced in the male reproductive tract by treatment with anti- androgens (which inhibit WD development) <26>. Similarly, Egf receptor is present at higher levels in the male than in the female reproductive tract at E18, and expression in females is increased after testosterone treatment <27>. Moreover, Egf in the absence of the testis, can maintain WD in culture and anti-Egf and anti-Egf receptor antibodies can prevent WD development in the presence of a testis <28, 27>. Interestingly, WD stabilisation by Egf can be blocked by anti-androgens, suggesting that the AR mediates Egf-induced effects <29>. This is supported by the finding that Egf can modulate AR activity in mesenchymal cells of the mouse foetal reproductive tract <30>.

Growth hormone (GH) has also been found in the foetal mouse male reproductive tract, and can stabilise the WD in culture <31>. Anti-GH antibody prevents WD stabilisation by the testis or testosterone in vitro. This effect can be reversed by supplementation with GH or, more effectively, with insulin-like growth factor 1 (Igf1), suggesting the effects of GH in the WD may be mediated by Igf1 <31>.

Igf has been detected in prepubertal mice in the myofibroblastic cells surrounding the epididymis and the type 1 Igf receptor is expressed in the epithelium <32>. An important role for Igf in WD development is further supported by the observation that Igf1 null mice have severe reproductive abnormalities <32>. They have a disproportionately small corpus and cauda epididymis, vas deferens and seminal vesicles and the cauda epididymis lacks the numerous ductal convolutions that are characteristic of this region <32>. These changes may be due partly to decreased testosterone levels in Igf1 null mice.

Transgenic mice that ectopically express fibroblast growth factor 3 (Fgf3) in the WD and prostate, have extremely enlarged reproductive tracts; in mice older than 4 months, WD derivatives weigh as much as 10% of total body weight <33>. The epididymides and vasa deferentia of these animals show epithelial stratification and contain haemorrhagic cysts. The physiological role of Fgf3 in the WD is not clear, but Fgf10 has been detected in the normal epididymis and is important for the development of seminal vesicles <34>. Similarly, Fgf7 is expressed in the developing mouse seminal vesicle in vivoand is thought to partially mediate androgen-induced growth <35>.

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Transforming growth factor β(Tgfβ) may also play a role in WD development. Tgfβ2 knockout mice have urogenital defects. Males examined by Sanford et al. <36> had ectopic testes and one mouse had unilateral testicular hypoplasia with lack of an epididymis and dysgenesis of the vas deferens. Tgfβreceptor type 3 null mice, which show decreased sensitivity to Tgfβ2, are subfertile when they survive until after birth, although it is unclear what is the direct cause of reduced fertility <37>.

AR may directly regulate expression of the growth factors discussed above, but may also indirectly regulate growth factor activity through other molecules. Connective tissue growth factor, e.g., which increases activity of Fgfs <38>, and Igf-binding proteins 2 and 6, which affect Igf activity, are upregulated during WD development in the rat <39> (fig. 4). Similarly, expression of proteins known to potentiate Tgfβactivity, such as dermatopontin <40>, increases in the developing rat WD <39>.