This study investigated the expression of steroid receptors in the murine developing mesonephros from E12.5 to E18.5. Using 3-D reconstruction, it was possible to compare distal versus proximal MTs in relationship to the gonad and WD. Specific expression patterns were revealed in the embryonic tubules that will form the mature efferent ductules, which develop from MTs and the cranial portion of WD (Table 2 summarizes the results for AR, ESR1 and PGR). Key findings in the present study are the following: 1) AR is the first steroid receptor to appear in MT and WD epithelium and is first expressed in MTs on the gonadal side; 2) epithelial expressions of ESR1 are specific to the connected MTs and cranial WD (efferent ducts); 3) rete cells express AR but not ESR1 and PGR before birth; 4) ESR1 is expressed in mesenchymal cells around the WD, especially in the caudal region; 5) AR and ESR1 are also expressed in mesenchymal cells around the testis artery; 6) PGR showed weak but specific expressions in the MT and cranial WD; 7) epithelial cells of the efferent ducts (derivatives of the MT and cranial WD) were the only site to express all three steroid receptors in the same epithelial cell.
AR signaling is necessary for the maintenance of the WD. Blockage of AR signaling perturbed the development of the WD, while activation of AR signaling induced persistence of the WD even in the genetically female mesonephros (Welsh et al. 2009; Jia and Zhao 2022). Surprisingly though, AR signaling for WD maintenance is mesenchymal and not epithelial, even with very strong expression of AR in the WD epithelium beginning at E18.5. Signaling from the WD mesenchyme to the epithelium was found to be crucial through the use of conditional AR knockout specifically in the WD epithelium, which showed normal epithelial cell differentiation in the epididymis but loss of basal cell differentiation (Murashima et al. 2011). Expression patterns of AR in the WD obtained by the present study are consistent with previous studies (Bentvelsen et al. 1995; Murashima et al. 2011).
In MTs, AR expression began as early as E12.5, although not in all epithelial cells. This discovery pushes the onset of AR expression earlier than previously reported, probably due to the difficulty in identifying MTs before E14 (Cooke et al. 1991). Most recently, there is a report in which AR expression is observed in the MTs even at E11.5 (Aksel et al. 2022). The present study, however, was able to trace MTs with 3-D reconstruction to localize AR at an earlier fetal age. AR showed more intense immunostaining at E15.5 and onward, indicating that the MTs are capable of responding to androgen after the gonadal fate is determined. Because the ESR1 expression in the MTs was faint during the earliest period examined, it is likely that androgen signaling is the dominant hormone for very early MT’s development and preceding the intense expression of ESR1 by several days.
Of special note, epithelial AR expression in the MTs was first more prominent near the gonad at E12.5, with intense expression spreading to the entire MTs and the cranial portion of the WD (common ductule) by E15.5. The caudal region of the WD showed weak staining for AR until E18.5. Previous studies reported that androstenedione, one of androgens, is capable of binding to AR (Jasuja et al. 2005) and can be secreted by fetal Leydig cells from E12.5 (Shima et al. 2013). Additionally, previous studies suggested that substances secreted from the gonad, such as androgens and anti-Müllerian hormone, would be dispersed in the gonadal and mesonephric mesenchyme of the cranial region and thus act first on the tubules and ducts in this region (Jost 1953; Tong et al. 1996; Yamamoto et al. 2018), which is consistent with the observed expression of AR in the developing mesonephros. Therefore, it is hypothesized that the development and maintenance of efferent ducts and the future epididymis begin with the MTs at the gonadal/rete junction by the action of androgen dispersed from the gonad. This hypothesis should be investigated in future studies using the MT-specific AR deletion model. Also, the dual presence of AR and ESR1 in the mesenchyme and epithelium of the cranial WD indicates early dependence on an androgen/estrogen balance for development of the future common efferent duct and maybe the initial segment of the epididymis (Hess et al. 2021). The potential role of ESR1 in maintaining MT connections and branching at the cranial WD region and rete testis should be investigated, as a previous study found a highly significant increase in the number of blind-ending efferent ducts in the ESR1-KO mouse (Guttroff et al. 1992; Hess et al. 2000).
The ESR1 expression was first observed in the mesenchyme and then later became strong in the epithelium of the MTs and cranial WD. The unique presence of ESR1 in MT epithelium provides a useful marker, beginning particularly around E15.5, for distinguishing the efferent ducts, including the common ductule, from the caudal WD (however, the initial segment epididymis could not be distinguished in this study). In contrast to the MTs, the rete epithelial cells were ESR1 negative at all ages studied, confirming a previous report in fetal mice and newborn mice and rat (Nielsen et al. 2000). However, the rete cells were AR positive as early as E15.5. Others have reported that post birth the rete testis epithelium of rats expresses both ESR1 and AR by postnatal day 4 and 5, respectively (Fisher et al. 1997; You and Sar 1998). While the presence and potential role of ESR1 in the rete testis remains unsettled for the developing male, there is good evidence for its epithelial expression in adult rodents (Fisher et al. 1997; Hess et al. 1997). Although there are few studies across species, one study reported human rete testis to be negative for ESR1 (Pelletier and El-Alfy 2000).
In the adult, a major function of rete testis chambers is the collection of sperm and seminiferous tubular fluids that are continuously produced in the testis (Free and Jaffe 1979; Free et al. 1980). In the present study, rete cells showed no evidence of ESR1 expression during development, which was surprising because the rete testis in ESR1-KO mice has cystic dilation that becomes massive in the adult (Eddy et al. 1996; Hess et al. 1997, 2021; Lee et al. 2000). Although the number of studies examining ESR1 expression in the rete epithelium are few, it has been assumed that this luminal dilation was due to fluid accumulation caused by dysfunction of the efferent ducts (Hess et al. 1997). Fluid from the rete testis flows rapidly into the several efferent ducts, whose epithelial function is to reabsorb nearly 90% of the luminal fluid, which produces an increase in the concentration of sperm and contributes to luminal flow from seminiferous tubules to the epididymal duct (Clulow et al. 1994; Kanazawa et al. 2022). Loss of ESR1 expression, as well as treatment with a potent anti-estrogen chemical, inhibits ion transport and water physiology in the efferent ducts, causing massive dilation of the ductules, as fluid cannot exit quickly enough through the single common duct that enters the head of the epididymis (Hess et al. 2000, 2021; Lee et al. 2000; Oliveira et al. 2002; Hess 2014).
In the present study, the rete testis lumen within the testis was not open at E18.5, but the region at the hilus where MTs join the rete cells was open; therefore, because the fetal rete testis cells were negative for ESR1, it is highly probable that rete testis dilation post birth in the ESR1-disruption models is likely due to the fluid back-up from the efferent ducts, rather than a direct induction of overgrowth and proliferation of the rete epithelium (Hess et al. 2021). The lumina of MTs began to open at E15.5, which would indicate that their luminal fluid is likely derived by epithelial secretions into the lumen, at least until the opening of the seminiferous tubular lumens into the rete testis, starting around postnatal day 10 in rodents (Lupien et al. 2006; Auharek and de França 2010). Expression of ESR1 in the MT epithelium did not become strong until E18.5, coinciding with the initial opening of the external rete testis, and thus may contribute to opening of the rete testis lumina until the post birth contribution of seminiferous tubular fluids. These data indicate that the MT epithelium during this early period of development has a secretory function, which would be consistent with data showing that cystic fibrosis transmembrane conductance regulator (CFTR) is upregulated in the absence of ESR1 (Toda et al. 2008). Further study of the MT physiology is warranted, in light of the observation that AR precedes the expression of ESR1. In cases of ESR1 disruption, it is possible that the rete testis epithelium post birth will not be able to accommodate the excess fluid from the efferent ductules (Hess et al. 1997), which could result in thinning of the epithelium and cystic expansion into the testis (see reviews: Hess, 2014 (Hess 2014), Hess et al., 2021 (Hess et al. 2021)).
It is now clear that ESR1 expression in MTs is essential for normal development of the efferent ducts and physiological maintenance of the luminal fluids, as well as normal development and adult response of the rete testis to these fluids. However, it is not clear how much of the fetal development is dependent on AR versus ESR1. Disruption of ESR1 function induces cystic dilation and abnormal development of both rete testis and efferent ducts. Treatment with high doses of an estrogen during fetal and neonatal development in rodents will also produce a nearly identical cystic dilation (Aceitero et al. 1998; Fisher et al. 1998, 1999; Rivas et al. 2002, 2003; Naito et al. 2014). Interestingly, it was discovered that neonatal high doses of estrogen caused a down-regulation of AR, without affecting ESR1 expression (McKinnell et al. 2001) and that co-treatment or subsequent treatment of an androgen with estrogen neonatally reversed the estrogen effect on AR and rete testis dilation (Rivas et al. 2003). Thus, there appears to be a unique dual regulation of this region in the male reproductive tract through both steroid receptors, suggesting that a balance in androgen/estrogen receptor signaling is necessary for normal development and function (Hess et al. 2021).
An overdose of estrogen during the neonatal period also induces inflammation in the efferent ducts, the epididymis, and vas deferens after puberty (Naito et al. 2014). Inflammation was found in the epididymis and vas deferens at an earlier age, and therefore, the neonatal exposure to estrogen probably affects the caudal part of the WD. In the present study, the epithelial expression of ESR1 was found in the MTs and cranial WD (common efferent duct) at E18.5, just before birth. The epithelium of the MTs and WD was surrounded by the condensed mesenchyme, and the mesenchymal cells showed positive for ESR1, especially in the caudal portion. These results suggest that the overdose of estrogen during the neonatal period did not affect the epithelium but rather the mesenchyme around the caudal epididymal duct. Furthermore, neonatal exposure to diethylstilbestrol, one of the estrogenic chemicals, affected the development of basal cells in the vas deferens and induced disruption of the epithelium, which was rescued by testosterone (Atanassova et al. 2005). Therefore, the mesenchyme-epithelium interaction, which needs an appropriate balance of estrogen and androgen signaling (Sipilä and Björkgren 2016), is probably essential for the basal cell development in the epididymis and vas deferens. In human, although epithelial cells in the efferent ductules and caput epididymis expressed ESR1, the mesenchymal expression of ESR1 was weak or negative during the fetal and adult period (Sullivan et al. 2019; Leir et al. 2020; Cunha et al. 2021), suggesting that the interaction between the mesenchyme and epithelium via ESR1 signaling is dispensable in human epididymis.
AR and ESR1 were found in the mesenchymal cells around the testicular artery but not in the epididymal artery in the present study. The mammalian testicular artery is winding and surrounded by the pampiniform plexus. This characteristic structure helps keep the testicular temperature lower than the body temperature (Harrison and Weiner 1949). However, it is unclear by what mechanisms the testis artery winds and the veins surround the artery during the development. To form the winding vessel, the structure around the endothelial cells is crucial. For instance, looping of the intestine is induced by, e.g., the different growth rate between the intestine and mesentery and the left-right asymmetry of extracellular matrix within the mesentery (Savin et al. 2011; Nerurkar et al. 2017). Future study should focus on the role that sex steroids might play in this aspect of vascular development.
PGR expression was found in the male MTs and common duct after E15.5, similar to human fetal tissues after 21 weeks (Magers et al. 2016). In the adult, PGR is enriched in selective cells of human efferent ducts (Ergün et al. 1997; Légaré and Sullivan 2020), limited expression in the cauda epididymis of the matured rat (Adebayo et al. 2017), but throughout the reproductive tract of adult quail (Nishizawa et al. 2002). Even though the receptor has been observed, there has been limited consideration of progesterone’s function during development of the male reproductive tract. In the present study, epithelial expression was similar to ESR1, although PGR was not found at the tip of the MTs. Unlike ESR1 staining, PGR was not expressed in the mesenchymal cells around the tubules. Although PGR was found with moderate expression at E18.5, a previous study reported no detection of PGR in the control rat male reproductive tract from postnatal day 18–90, except for the parasympathetic ganglia of the prostate (Williams et al. 2000). However, neonatal exposure to estrogen induced a transient expression of PGR in the mesenchyme of the epididymis (Williams et al. 2000).
Progesterone in high concentrations in the mother’s serum is transferred into the fetus. The fetal liver can metabolize progesterone, but its ability is reported in sheep to be lower in the male than in the female (Siemienowicz et al. 2020). Therefore, it is not surprising to find that progesterone administration to the female sheep during early pregnancy upregulated several fetal testicular genes, although the effects may have been due to negative effects on the pituitary (Siemienowicz et al. 2020). However, treatment with progesterone post-birth apparently did not show any effects on testis, epididymis or prostate (Jones 1980) and male PGR knockout mice are fertile, although having reduced sexual behaviors (Lydon et al. 1995; Phelps et al. 1998; Schneider et al. 2005; Yang et al. 2013). Thus, the role of progesterone in the developing male tract remains an enigma.
In conclusion, 3-D reconstruction revealed more accurately the expression patterns for three steroid hormone receptors in the developing male reproductive tract, with a special focus on the mesonephros, where ESR1 plays a unique role in the function of efferent ducts (Hess 2014; Hess and Cooke 2018; Hess et al. 2021). AR was clearly the dominant receptor throughout the male tract, both in the mesenchyme and epithelial cells, with early expression at E12.5 in the MT. ESR1 was found at E12.5 but mostly in the mesenchyme surrounding MT near the cranial WD and did not show strong epithelial immunostaining until E18.5. Epithelial expression of ESR1 was specific to the MTs (branched efferent ducts) and cranial WD (common efferent duct), which indicates that its participation in the regulation of fluid resorption and epithelial function in the efferent ducts begins just before birth, which is consistent with reports from ESR1 knockout mouse phenotypes. The mesenchymal dominance of ESR1 throughout the developing tract helps to explain the induction of a massive inflammatory response over time following neonatal treatment with estrogen (Naito et al. 2014). However, the absence of ESR1 in the fetal rete testis cells raises numerous questions regarding their cystic response to loss of ESR1 or estrogen treatment in utero and in the neonate. Finally, most studies focus on one hormone receptor at a time, but future studies are required to better understand the overlapping roles that three steroid receptors (AR, ESR1, and PGR) would have when present together in the same MT epithelial cell. Hopefully, these data will help in the design of studies for the treatment of male infertility caused by dysfunction in the head of the epididymis. Recent studies reported an advancement in the use of male reproductive tract ex vivo methods (Hasegawa et al. 2020; Jia and Zhao 2022; Inoue et al. 2022), which could be used in future investigations of steroid regulation of the developmental process.