Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.


Introduction
Efferent ducts in man form the bulk of what is called the head or proximal part of the epididymis (Sullivan et al. 2019;Légaré and Sullivan 2020), and pathological changes are more commonly found in this region in larger mammals, including obstructive lesions, which contribute substantially to male infertility (Ball and Mitchinson 1984;McEntee 1990;Rajalakshmi et al. 1990;Pal et al. 2006). The high incidence of epididymal disjunctions associated with epididymal anomalies points to the importance of understanding the endocrine regulation for development of the male ductal systems (Logsdon et al. 2022).
Efferent ducts originate from mesonephric tubules (MTs) and epididymis from the Wolffian duct (WD). Because these tubular systems including connection between rete cells and MTs develop in both sexes (Omotehara et al. 2020), their maintenance and development require steroid hormonal signaling, with testicular androgen secretion being essential in the male (Welsh et al. 2009;Jia and Zhao 2022). In the female, lack of androgen is thought to result in regression of MTs and WD, but remnants, called ductuli efferentes ovarii or epoöphoron, are still present in the adult. A previous study showed that androgen receptor (AR) in the epithelium is not necessary for maintenance and development of the WD, but instead it is the AR-expressing mesenchymal cells that mediate androgen signaling to the WD epithelium (Murashima et al. 2011). However, AP2ɑ-Cre mice used in the previous study were reported to be insufficient for eliminating the target gene in MTs (Kitagaki et al. 2011). Therefore, the function of AR, which is also expressed in epithelium of the MTs, at least in the later stage (Hess et al. 2021), remains unclear.
Estrogen is traditionally considered to be a female sex steroid, but Sharpe and Shakkebaek (1993) reported the "oestrogen hypothesis" suggesting that appropriate estrogen signaling is crucial for development of the male reproductive tract (Sharpe and Skakkebaek 1993). Deletion of ESR1 resulted in dilation of rete testis and efferent ducts in the adult (Hess et al. 1997) and even in postnatal day 6-10 mice (Lee et al. 2000(Lee et al. , 2009Oliveira et al. 2002;Cho Hess et al. 2021), indicating that absorption of luminal fluid by the efferent duct epithelial cells requires ESR1 signaling from the neonatal period. On the other hand, neonatal exposure to estrogen or estrogenic chemicals, such as diethylstilbestrol or bisphenol A, induces abnormalities in the reproductive tracts after puberty, such as epididymal inflammation and dilation of the rete testis and efferent ducts (vom Saal et al. 1998;Rivas et al. 2002;Atanassova et al. 2005;Naito et al. 2014). However, detail of how neonatal estrogen exposure influences the male reproductive tracts after puberty remains incomplete, although it was shown that neonatal estrogen down-regulated the tissue AR (McKinnell et al. 2001;Rivas et al. 2002Rivas et al. , 2003. Although some previous studies examined the expression patterns of these steroid receptors, the results differ, possibly due to the use of different detection methods, e.g., autoradiography or immunohistochemistry (Hess et al. 2021). Additionally, using only two-dimensional observations resulted in difficulties recognizing the type of tubule having a positive reaction. Distinguishing MTs, the common duct (a cranial portion of the WD), and caudal WD in histological sections is difficult (Omotehara et al. 2022a); however, three-dimensional (3-D) analysis provides the necessary resolution and imaging for determining more precisely the location of sex steroid receptors in the male reproductive tract. Our previous use of 3-D analysis revealed the unique contact between developing MTs and rete testis and discovered that the common efferent duct is derived from the cranial region of WD (Nakata et al. 2021;Omotehara et al. 2022a, b). Therefore, in the present study a more precise localization of major sex steroid Fig. 2 Representative pictures of the reconstructed tubules and photos of estrogen receptor (ESR1) expression at E12.5. a Reconstructed mesonephric tubules (blueish and greenish colors) and Wolffian duct (reddish line) of the left mesonephros are illustrated. The top of the panel is the cranial side. The red line indicates the cranial region of the Wolffian duct (Cra-WD), from which each mesonephric tubule (MT) branches, and the pale red line represents the Wolffian duct (Caud-WD) located at the caudal region from the most caudal branching point of the MT. b A representative photo of ESR1 localization. The region surrounded by boxes is shown in (c) and (d). c The magnified photo shows the mesothelium (arrows) and mesenchyme around the Müllerian duct (MD). d The photo displays the same section where Collagen type IV is detected, with an indication of the associated tubules for the reconstructed lines. The regions surrounded by boxes are magnified in (e) and (f). e The magnified photo of the cranial Wolffian duct and the surrounding mesenchymal cells. f The magnified photo of a mesonephric tubule (MT), with slight expression of ESR1. Arrowheads indicate positive epithelial cells (the color corresponds to the color in the reconstructed tubule). Bars = 100 µm (b), 50 µm (d), 10 µm (c, e, f) 1 3 receptors, such as AR, ESR1, and progesterone receptor (PGR) was determined in the mesonephros and surrounding tissues, which will provide a basis for future studies of steroid hormonal regulation of development in the male reproductive tract.

Animals
This study was approved by the Tokyo Medical University Committee (Permission #R3-0066). C57BL/6 J mice were purchased from Japan SLC,Inc. and maintained at a specific pathogen-free facility at Tokyo Medical University. They were kept at 22-24 degC and 50%-60% relative humidity with a 12-h light-dark cycle. Male and female mice were mated for one night, and vaginal plug presence was checked the following morning. The date of plug confirmation was designated as E0.5.

Histology
The gonad-mesonephros complex was collected from the mouse fetus at E 12.5, 15.5, and 18.5 and embedded in paraffin, as previously reported (Omotehara et al. 2022b). The paraffin blocks were serially cut at 5-µm-thick and placed sequentially on 10 slides, with 50-µm-interval sections per slide. The tissues from three males were analyzed at each developmental stage. Immunohistochemistry was performed as in the previous report (Omotehara et al. 2022b). The primary antibodies utilized are listed in Table 1. Details of the anti-AR rabbit antibody are described elsewhere (Prins et al. 1991;Vornberger et al. 1994). Validation for anti-ESR1 antibody was provided by the manufacturer. Western blot was performed to confirm anti-PGR antibody (Fig. S1). EnVision rabbit and EnVision mouse for DAB (Agilent Dako) were used as secondary antibodies for rabbit and mouse antibodies, respectively. The reacted antibodies were detected with 3-amino-9-ethylcarbazole (AEC substrate kit, Peroxidase (HRP), SK-4200, Vector), and Gill's hematoxylin V (Muto Pure Chemicals) was used for counterstain. The sections were digitized with a virtual slide scanner, Panoramic MIDI II (3DHISTECH), at × 20 magnification. After the reacted antibodies were eliminated by autoclave at 121 degC for 20 min in 10 mM citrate buffer, pH6.0, the immunohistochemical procedures following the protein blocking were repeated to detect Collagen type IV in all sections.

3-D reconstruction
At each stage, the tissues from three male individuals were analyzed with Amira software (version 2020.2, Thermo Fisher Scientific), as in the previous study (Omotehara et al. 2022a). Briefly, the serial sections on which Collagen type IV was detected were aligned. After the MTs and WD were segmented and reconstructed, the core lines running through the center of the reconstructed tubules were drawn in the software.

E12.5
The 3-D structure of MTs in E12.5 (Fig. 1a) was similar to that of E11.5 shown in a previous study (Omotehara et al. 2022a). The MTs were folded twice between the gonad and WD. Some tubules showed connection with the WD, while others disconnected (as would be observed for tubules that would be expected to regress in the more caudal region). Therefore, the WD could be divided into two portions; the cranial WD (Cra-WD) that divided the MTs and a caudal WD (Caud-WD) contiguous to the urethra. Positive reactions of AR were found in mesenchymal cells around the WD, except for mesenchyme that surrounded the Müllerian duct, consistent with the previous study that reported AR expression in the mesenchyme around the WD as early as E13 (Cooke et al. 1991;Murashima et al. 2011) (Fig. 1b-d). This study found the AR-positive mesenchymal cells localized especially in the cranial portion of shown in (c)-(e). c The photo displays the same section where Collagen type IV is detected. The dotted line indicates the rete testis region (RT). d The photo magnifies the rete region shown in (b). An arrowhead indicates the Sertoli cell. e The magnified photo of the disconnected MT. f Another representative photo of AR localization to illustrate Cra-versus Caud-WD. The region surrounded by a box is shown in (g)-(i). g The photo displays the same section where Collagen type IV is detected. h The magnified photo of the Cra-WD and Caud-WD, respectively, with AR reactions. i The magnified photo of the Caud-WD in the more caudal region with AR reactions. Bars = 100 µm (b, f), 50 µm (c, d), 10 µm (e, g-i) 1 3 the WD. The epithelial expression of AR was also found, and with 3-D analysis such tubules were identified as the MTs (Fig. 1b-d). Notably, the reaction for AR was different between the sections even of the same MT. Because a single MT was long and therefore folded in the mesonephros, a serial MT could be divided into the regions near the WD or rete cells. We found that the AR-positive epithelial cells were present in the MT region near the junction with rete cells, regardless of their connection to the WD (Fig. 1). However, epithelial cells in the MTs neighboring the rete cells and rete cells themselves were negative for AR (Fig. 1d). ESR1 was detected in the mesenchyme around the WD, especially in the cranial region, but the mesenchyme of the Müllerian duct was mostly negative (Fig. 2a-e). The mesothelium that covered the Müllerian duct was positive for ESR1, but the Müllerian duct epithelium was negative ( Fig. 2c). In the MTs, a faint positive reaction for ESR1 was found in the middle portion of some MTs (Fig. 2f).
The positive reactions for PGR were found around the blood vessels, but these reactions may be falsely positive because the reactions were observed in the extracellular region. Therefore, PGR was not expressed in the gonads and mesonephros at E12.5 (Fig. 3).

E15.5
At the middle period of the gonadal development, E15.5, 3-D analysis confirmed that the cranial portion of the WD to be folded and that the MTs were less convoluted in structure than at E12.5 (Fig. 4a), similar to a previous study (Omotehara et al. 2022a). The mesenchymal expression of AR was strong and spread broadly in the mesonephros and testis (Fig. 4b). The MTs that were connected with the WD showed strong epithelial reactions for AR along the whole length of the tubules (Fig. 4c, d). On the other hand, the MTs that were not connected with the WD possessed vast lumen, and their epithelium was thin with weak reactions for AR (Fig. 4e). The rete cells also showed weak expression of AR, although testis cords (Sertoli and germ cells) were negative ( Fig. 4b-d). The epithelial cells of the Cra-WD were strongly positive for AR ( Fig. 4f-h). AR was also found in the Caud-WD, but the reactions were weaker than in the Cra-WD (Fig. 4h, i).
Although having weak staining for ESR1, the positive epithelial cells in the MT region near the Cra-WD had a stronger reaction than at E12.5 ( Fig. 5a-d). Rete cells and the region of the MTs adjacent to the rete cells were negative for ESR1 (Fig. 5e). The Cra-WD, from which the MTs branched, was also positive for ESR1, but the Caud-WD was not ( Fig. 5f-j). The mesenchymal expression of ESR1 was spread throughout the mesonephros and testis (Fig. 5b, f). Nortably, ESR1-positive mesenchymal cells were more condensed around the Caud-WD (Fig. 5f).
Positive reactions for PGR were found in some epithelial cells at E15.5. Three-dimensional analysis revealed these cells to be located in MTs near the Cra-WD ( Fig. 6a-d). Rete cells and epithelial cells of the MT region near the junction with the rete cells were negative for PGR (Fig. 6c, d). The Cra-WD was also positive, clearly showing the border of the Cra-WD (future common efferent duct) and Caud-WD (future epididymis) ( Fig. 6e-g). The positive reactions were also found around the blood vessels, but these reactions may be falsely positive because the reactions were observed in the extracellular region.

E18.5
The MTs and WD were highly coiled in the reconstructed structures, similar to those expected in mature efferent ducts and the epididymal duct (Fig. 7a, b). As shown in the previous study (Omotehara et al. 2022a), each MT branched from a single common ductule (Cra-WD). AR was strongly positive throughout the tubules of the mesonephros, including the connected MTs, the Cra-and Caud-WD (Fig. 7). In contrast, the rete cells were weakly positive, showing a clear border between the MT and rete testis (Fig. 7d, e). Most mesenchymal cells in the mesonephros (epididymis) were also positive for AR, and the tubules and testicular artery/ vein were surrounded by these condensed mesenchymal cells (Fig. 7c-f). AR-positive cells were also observed in the interstitium of the testis, but Leydig cells, which had a larger nucleus and greater amount of cytoplasm than other interstitial cells, were negative or weakly positive for AR (Fig. 7g, h). Sertoli cells were also negative for AR, in contrast to the rete cells with weakly positive reactions (Fig. 7e,  g, h). The epithelium of both the Cra-and Caud-WD showed strong positive reactions for AR (Fig. 7g, i-m). However, MTs without contact with the WD (more caudal regressing MTs) showed weakly positive or negative staining for AR (Fig. 7n). AR-positive mesenchymal cells were less around the epididymal artery and vein than in the testicular artery and vein (Fig. 7f, o).
Epithelial expression of ESR1 in the MTs was stronger in E18.5 than in E15.5 (Fig. 8a-e). The rete cells were negative for ESR1, which resulted in a clear demarcation between the rete cells (negative) and MT (positive) (Fig. 8c-e), as shown in previous studies (Nielsen et al. 2000;Sar and Welsch 2000). The mesenchymal cells around the MTs and rete cells were negative, but those around the testicular artery and vein were positive, although the number of the positive cells for ESR1 were smaller than those for AR (Figs. 7f and 8f). The ESR1-positive mesenchymal cells were mainly found around the WD, especially the most caudal region (Fig. 8g). ESR1 expression was also found in ◂ the interstitial cells in the testis, including the Leydig cells, but not in the Sertoli cells (Fig. 8g, h). The Cra-WD was positive for ESR1 like the MTs (Fig. 8i, j). In this study, we defined the Caud-WD as the region of the WD caudal to the most caudal branching point of the MT, but the proximal region of the Caud-WD (following the Cra-WD) showed positive for ESR1, similar to the Cra-WD (Fig. 8g-m). The caudal MTs without contact with the WD were negative or weakly positive for ESR1 (Fig. 8n). The mesenchymal cells around the epididymal artery and vein were also negative, in contrast to those around the testicular artery and vein (Figs. 7o and 8o).

3
PGR expression was not found in both the rete cells and a tip of the MTs (Fig. 9a-e). However, the Cra-WD and MT region near the Cra-WD showed PGR positive (Fig. 9f-h). The proximal region of the Caud-WD (following the Cra-WD) was also positive for PGR similar to the expression pattern of ESR1 (Figs. 8l, m and 9i-k). Mesenchymal cells in the mesonephros and testis, including the rete cells, were also negative for PGR unlike AR and ESR1 (Fig. 9c-k).

Discussion
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 examine the expression patterns with precise identification of each region of the MT and WD for the first time. These results are summarized in Table 2. 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 the MT region near the junction with rete cells; 2) epithelial expressions of ESR1 are specific to the connected MTs and Cra-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 Cra-WD; 7) epithelial cells of the efferent ducts (derivatives of the MT and Cra-WD) were the only site to express all three steroid receptors in the same epithelial cell.
Expression patterns of AR in the WD were reported in previous studies (Bentvelsen et al. 1995;Murashima et al. 2011). Especially in Murashima et al. (2011), stronger expression of AR in the WD in the cranial portion of the mesonephros was reported, and this study confirmed the results of these studies. However, these previous studies did not focus on the more cranial region, including MTs and Cra-WD, where we found AR expressions. In human efferent ducts, AR expressions were found by 7 weeks of gestation and were strengthened by 12 weeks of gestation (Shapiro et al. 2005), similar to findings from mice in this study. Most recently, a report in which AR expression was observed in the MTs even at E11.5 (Aksel et al. 2022) pushed the onset of AR expression earlier than previously reported (Cooke et al. 1991). We also found AR expression in the MTs as early as E12.5, although not in all epithelial cells. 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). Because this study showed that the ESR1 expression in the MTs was faint at E12.5, it is likely that androgen signaling is the dominant hormone for very early MT's development. Consistent with this idea, the AR expressions in human efferent ducts also preceded testosterone production (about 8 weeks of gestation onward in fetal testes (Reyes et al. 1973;Siiteri and Wilson 1974)) and ESR1 expression (by 12 weeks of gestation (Cunha et al. 2021)).
Of special note, epithelial AR expression was first more prominent in the MT region near the junction with rete cells at E12.5, with intense expression spreading to the entire MTs and the Cra-WD (common ductule) by E15.5. The Caud-WD did not become strong staining for AR until E18.5. 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). A previous study suggested that AR signaling mediated via the mesenchymal cells around the WD was necessary for the maintenance and development of the epididymis (Murashima et al. 2011). However, at least in E12.5, AR expression was more intense in the MTs than in the mesenchyme around the WD in this study. 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 MT-specific AR deletion models.
The ESR1 expression was first observed in the mesenchyme and then later became strong in the epithelium of the MTs and Cra-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 epididymal duct. The potential role of ESR1 in morphogenesis of these tubules remains unclear, but investigation on maintaining MT-rete testis connections and branching at the cranial WD region and rete testis is necessary, as a previous study found a highly  (Guttroff et al. 1992;Hess et al. 2000).
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. , 2021Lee et al. 2000;Oliveira et al. 2002;Hess 2014). Although these suggestions were obtained in the adult mice, strong ESR1 expression in the fetal MTs and Cra-WD and phenotype of the ESR1-KO mice suggest that secretion and reabsorption of the luminal fluid occur in the MTs and Cra-WD (efferent ducts) and that the reabsorption is mediated via ESR1 signaling even at the fetal period.
In contrast to the MTs, the rete epithelial cells were AR positive but ESR1 negative at all ages studied, confirming a previous report in fetal mice and human and newborn mice and rat (Nielsen et al. 2000;Magers et al. 2016). Others have reported that 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). There is also good evidence for epithelial expression of ESR1 in adult rodents (Fisher et al. 1997;Hess et al. 1997). Although there are few studies across species, one study reported adult 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. 1997Hess et al. , 2021Lee et al. 2000). Although the extent of the contribution of ESR1 to absorbing luminal fluid in the rete testis is unclear, negative staining for ESR1 in the fetal rete testis suggests 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 as assumed previously (Hess et al. 1997(Hess et al. , 2021Hess 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. 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.
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. On the other hand, AR expression preceded ESR1 expression in the fetal rete testis, but it is not clear how much of its development is dependent on AR versus ESR1. It was discovered that neonatal high doses of an exogenous estrogen, diethylstilbestrol, 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 (McKinnell et al. 2001;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). Further study of the morphogenesis and physiology of the rete testis is warranted, in light of the observation that AR precedes the expression of ESR1. Treatment with high doses of an estrogen during fetal and neonatal development in rodents will produce a nearly identical cystic dilation of the rete testis (Aceitero et al. 1998;Fisher et al. 1998Fisher et al. , 1999Rivas et al. 2002Rivas et al. , 2003Naito et al. 2014). Furthermore, 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. This delayed effect is probably due to the incomplete barrier of the epididymal epithelium, which allows immune cell infiltration and immune response to the spermatozoa in the lumina after the onset of spermatogenesis in the testis. In the present study, the epithelial expression of ESR1 was found in the MTs and Cra-WD (common efferent duct) at E18.5, just before birth. On the other hand, the condensed mesenchymal cells surrounding the WD showed positive for ESR1, especially in the caudal portion of the mesonephros. These results suggest that the overdose of estrogen during the neonatal period did not affect the epithelium but rather the mesenchyme around the epididymal duct in the caudal portion. 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), indicating the mesenchyme-epithelium interaction, which needs an appropriate balance of estrogen and androgen signaling (Sipilä and Björkgren 2016) in the epididymis and vas deferens. Therefore, the overdose of estrogen probably affects the mesenchyme-epithelium interaction to inhibit development of the epithelial barrier, such as tight junction. 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 or weak in human epididymis. Future studies about how the mesenchyme contributes to the epididymal development are necessary with focusing on the species differences.
The mammalian testicular artery and vein show characteristic structure. The testicular artery winds well, and the testicular veins surround the artery in a reticular pattern and are called the pampiniform plexus. These characteristic structures help 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. 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). Therefore, to form the winding vessel, the structure around the endothelial cells is crucial. In this study, AR and ESR1 were found in the mesenchymal cells around the testicular artery but not in the epididymal artery. These results suggest that the AR and/or ESR1 signaling in the mesenchymal cells around the testicular artery and vein play a role in promoting cell proliferation and production of extracellular matrix to form the characteristic structure of the testicular artery and vein systems. 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 Cra-WD 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 in the MTs near the junction with the rete testis. 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 1 3 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 showed only moderate effects on testis, epididymis or prostate, compared to the estrogen effects (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 near the junction with the rete cells. Epithelial expression of ESR1 was specific to the MTs (branched efferent ducts) and Cra-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.