Animal experiments were carried out at our local animal facility, with ethical approval from the national Animal Experiments Inspectorate (license number, 2020-15-0201-00539) and overseen by the local Animal Welfare Committee. The animal studies were carried out in accordance with the EU Directive, 2010/63 for the protection of animals used for scientific purposes.

Time-mated Sprague-Dawley rats (Crl: CD(SD), RRID: RGD_734476), 8–9 weeks (210–260 g; Charles River Laboratories, Sulzfeld, Germany) were used, with gestational day (GD) 1 defined as the day following overnight mating. All animals were housed in High Temperature polysulfone (H-TempTM) cages (Tecniplast, Buguggiate, Italy) with wood chip bedding, nesting material, and a Tapvei wooden shelter (Brogaarden, Lynge, Denmark). They were kept in a controlled environment with 12 h light/dark cycles, 55 ± 10% humidity, 22 ± 5 °C temperature, and 75 air changes per hour. The animals were fed a standard soy and alfalfa-free diet based on Altromin, 1314 (Altromin GmbH, Lage, Germany) and provided tap water in bisphenol A-free bottles (polyphenylsulfone 400 ml, 84ACBT0402PFS, Ultem, SCANBUR, Karlslunde, Denmark) ad libitum. All dams were housed in pairs until GD17 and single-housed hereafter until the end of the experiment.

Rat fetuses were collected daily from time-mated control dams at developmental stages GD15 to GD21 (n=1 dam per stage for morphological characterization by H&E stain, and n=3 dams per stage for RNAscope analyses). Dams were sedated in CO₂/O₂, decapitated, and fetuses were collected. The sex of the fetus was determined by gonadal inspection under a stereomicroscope (GD16, 17, 18, 20, and 21) or by genotyping of fetal rat tail for the SRY gene (GD15, and GD19). Whole fetuses or lower bodies were then fixed in 10% formalin, and later dehydrated in an Excelsior AS tissue processor (Axlab, Vedbaek, Denmark) and embedded on their side in paraffin.

Flutamide (>99% purity, CAS # 13311-84-7) was purchased from Merck Life Science (cat. # F9397, lot # MKCL6929). Corn oil (cat. # C8267; Merck Life Science, Søborg, Denmark) was used as vehicle. The doses were determined based on previous rat toxicity studies using flutamide as a model chemical and reporting on hypospadias (22, 23). The flutamide solutions were stored at room temperature, protected from light, and under constant stirring during the experiment. The study design is illustrated in Figure 1 . For exposure study 1, postnatal examinations, time-mated dams were delivered to the animal facilities on GD3. The day after arrival, dams were weighed and semi-randomly assigned to three exposure groups with similar body weight (bw) distributions: 0, 6, or 18 mg/kg bw/day flutamide (n=11–12 dams per group). Flutamide or vehicle (corn oil) was administered by oral gavage, using a dosing volume of 2 ml/kg bw, from GD7 to pup day (PD) 28 (with PD1=GD23), excluding the day of birth. Rat offspring were sexed and weighed the day after birth. This procedure was repeated on PD 6, 14, 22, and 28. On PD1, anogenital distance (AGD) was measured using a stereomicroscope with a micrometer eyepiece. On PD14, the number of nipples was registered in all live rats. AGD and nipples were registered by a trained technician blinded with respect to exposure group. Testicular descent, also blinded to exposure group, was evaluated in all male rats from PD14 until PD28. The day of testicular descend was registered, and rats without full testicular descent at PD28 were registered as either uni- or bilateral cryptorchid. One or two pups per litter, randomly selected, were dissected on PD6, 16, 22, and 28. On all dissection days, the selected rats were weighed and decapitated, under CO₂/O₂ anesthesia for the three latter ages. The external genitalia of male offspring selected for dissection were inspected under a stereomicroscope by a technician blinded with respect to exposure group, and the presence of genital malformations was registered. A scoring system was used on PD28 to classify the severity of hypospadias and genital feminization: Score 0: No malformations (normal penis with urethral opening at the tip). Score 1: Mild hypospadias (the urethral opening is placed on ventral side of penis at the distal end). Score 2: Moderate hypospadias (the urethral opening is placed approximately on the middle along the length of the penis). Score 3: Severe hypospadias (the urethral opening is placed at the base of the penis on the ventral side). Score 4: Feminized genitalia (the external genitalia appear completely feminized).

On all designated dissection days, the penises were isolated and weighed. On PD16, the testicles, epididymis, ventral prostate, seminal vesicles, and levator ani muscle were isolated and weighed. On PD28, the dams were sedated and killed by decapitation under CO₂/O₂ anesthesia. The number of implantations in the dams were counted, and their liver isolated and weighed.

For exposure study 2, prenatal examinations, time-mated dams were delivered to the animal facilities on GD4. The day after arrival, dams were weighed and randomly assigned to one of three exposure groups with similar bw distributions: 0, 3, or 6 mg/kg bw/day flutamide. Flutamide or vehicle (corn oil) was administered by oral gavage from GD7 until necropsy at GD17 (n=4-6), GD19 (n=4-7), and GD21 (n=7-8). On the day of necropsy, dams were exposed to flutamide 1 h ±15 min prior to sedation in CO₂/O₂ and subsequent decapitation. The number of implantations and fetuses were recorded. Fetuses were isolated, and their sex was determined by visual inspection of gonads during dissection under a stereomicroscope. On GD21, the fetuses were also weighed, and AGD was measured as described above. At all stages, lower bodies or whole fetuses were fixed in 10% formalin, or the GT was isolated and placed in RNAlater and stored at -80 °C. Formalin-fixed lower bodies and full bodies were dehydrated in an Excelsior AS tissue processor (Axlab) and embedded on their side in paraffin.

Micro-CT scanning was performed on paraffin-embedded fetuses from GD21 with a Phoenix Nanotom M (Waygate Technologies), operated using XS Control and Phoenix Datos|x acquisition and reconstruction software. Samples (n=3 fetuses from three different litters per group) were scanned for 10 min at 4-6 µm resolution collecting 1199 projections through a full 360-degree rotation. An X-ray tube voltage of 50 kV and current of 300 microAmp was used with a CVD coated tungsten target. Reconstructed volume data were processed using Avizo3D Pro (Thermo Fisher Scientific), reorienting specimens to common reference planes for transverse, sagittal and coronal cross-sections through the data. For 3D renders, the fetuses’ soft tissue was segmented from the µCT scan data using Avizo’s interactive thresholding tool to select slightly denser epithelial tissue and filling this in to create a sample mask. Animations illustrating the sagittal cross-section through the 3D render and in 2D are shown in Supplementary Material (see Supplementary Data Sheet 1 ). To highlight features, surface meshes of sample masks were generated in Avizo for ray-traced rendering.

Total RNA was extracted from GD17 and GD19 GTs using the RNeasy Micro kit (cat # 74004, Qiagen, Hilden, Germany) and from GD21 GTs using the RNeasy Mini kit (cat # 74106, Qiagen). The sample sizes for each stage were nine for control males, six for flutamide 3 mg/kg bw/day males, ten for flutamide 6 mg/kg bw/day males, and six for control females. In some cases, multiple (at max three) fetuses per litter were used for RNA extraction. Extractions were performed according to manufacturer’s protocol, including treatment with DNase (cat # 79254, Qiagen). cDNA was synthesized from 350 ng total RNA using the Omniscript RT kit (cat # 205113, Qiagen) and Random Primer Mix (cat # S1330S, New England Biolabs, Ipswich, MA, USA). For RT-qPCR, 3 µL 1:20 diluted cDNA was added to TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG (cat # 4352042, Applied Biosystems^®, Waltham, MA, USA) and a TaqMan™ Gene Expression Assay in a 384-well plate. A list of the gene assays is provided in Supplementary Table 1 . The RT-qPCR reactions were run on a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems^®). Relative gene expression was calculated using the comparative Ct method and the geometric mean of two references genes Rps18 and Sdha (24).

Lower section of bodies and whole bodies were sagittally sectioned at 5 µm thickness. Tissues were deparaffinized by heat treatment (60 °C for 30–60 min) and petroleum wash and subsequently rehydrated by ethanol treatment in decreasing strengths. For structural analyses (one fetus per examination stage), sections were stained with hematoxylin and eosin (H&E), dehydrated through an ethanol series, and mounted using Eukitt^® Quick-hardening Mounting Medium (cat # 03989, Merck Life science). Expression of Ar, Esr1 RNA molecules in the GT (minimum three litters per staining, one fetus per litter) was analyzed on the deparaffinized sections using the RNAscope^® in situ hybridization kit (cat # 322350 Bio-Techne, Dublin, Ireland) according to manufacturer’s instructions. Briefly, endogenous peroxidase activity was blocked with hydrogen peroxidase treatment, and sections were boiled in RNAscope^® Target Retrieval Buffer. A hydrophobic barrier pen (cat # 310018, Bio-Techne) was used to draw a barrier around each section, and the sections were left to dry overnight. The next day, sections were protease-treated for 15 min, and the probe of interest hybridized for 2 h at 40 °C. A negative and a positive control probe were also included. The probe signal was amplified by addition of 6 RNAscope^® amplification probes, and signal was detected by RNAscope^® Fast Red chromogenic substrate. The sections were counterstained in hematoxylin, dried at 60 °C for 30 min, and mounted in VectaMount^® Mounting Medium (cat # 321584, Bio-Techne). A list of the probes used is provided in Supplementary Table 1 . Sections were scanned using the PANNORAMIC Midi II slide scanner (3D Histech, Budapest, Hungary), using a 40x objective with 116x Native output resolution. Images were processed in CaseViewer version 2.4. For changes between groups in exposure study 2, semi-quantitative analyses were made in ImageJ version 2.14.0 using contrast thresholding.

All statistics were performed in R version 4.2.3, except for nipple/areola counts. AGD index (AGDi) was calculated as AGD/³√body weight. AGD, AGDi, and body weights were analyzed using a linear least square means mixed-effects model (lme4 (25), RRID: SCR_015654) with litter as a random and nested factor (except for PD16 and PD28 body weights, for which only 1 animal per litter was weighed). For birth weight, number of pups was included as a co-variate. P-values for exposure were obtained by Dunnett’s post hoc analysis. Dam and pregnancy data were analyzed by linear least square means regression and Dunnett’s post hoc analysis, with the exception for post-implantation and perinatal loss, for which the Kruskal-Wallis test and Dunn’s post hoc test were used. Reproductive organ weights were analyzed by linear least square means regression and Dunnett’s post hoc test, using body weight as a co-variate for the organ weights. qPCR data was analyzed using least square means regression and Dunnett’s post hoc analysis. For organs smaller than the detection limit of the scale (3 mg) the weight was imputed to 3 mg. In cases of organ agenesis (this was seen for prostate and seminal vesicles), the weight of the organ was set to 0 mg. Nipple retention was analyzed in SAS by generalized estimating equations (GEE) using a binomial model with 13 as the maximal number of nipples and using litter as the cluster. “Exchangeable” was used as the correlation structure, and significance level was assessed by a Hommel procedure. Data are presented as mean ± SD or boxplot with min, max, and 25^th, 50^th, and 75^th percentiles, *P<0.05, **P<0.01, ***P<0.001.

To gain a comprehensive overview of each stage of rat GT development, we mapped the development of the male rat GT between GD15–21 by histological assessments ( Figure 2 ). We combined terminologies from previous descriptions of rat (26–28) and mouse (29) GT development at selected time points, utilizing these to map the timing and appearance of specific structures of the rat GT. The GT arises from two swellings that merge around the cloaca, which by GD15 has separated into the urogenital sinus and hindgut by the urorectal septum. At this stage, a urethral plate has formed distally on the ventral side, and this will later generate the urethra ( Figure 2A ). At GD15 and GD16, the GT grows, and the dorsal mesenchyme expands ( Figure 2B ). From GD17, a dense, central penile mesenchyme is visible in the center of the GT, which continues to expand throughout fetal development to form most of the glans penis by GD21 ( Figures 2C-G ). At GD18, the proximal urethra has formed, and the urorectal duct is seen as an opening of the urethra. In males, the urorectal duct is closed by the differentiation of the proximal part of the urethra ( Figure 2D ). This differentiation is largely driven by the urorectal septum, which invades the urethral plate from ~GD18 to form an internalized urethra in the glans ( Figures 2D-G ). In parallel, prepuce is visible on the dorsal side of the GT from GD18 and on the ventral side from GD19 ( Figures 2D, E ), and the prepuce gradually envelops the glans in a proximal to distal direction from GD18 to GD21, fusing at the ventral midline ( Figures 2D-G ).

Using in situ RNA hybridization (RNAscope^®), we mapped the transcriptional distribution of Ar and Esr1 in the GT from GD15-21 ( Figure 3 ). Ar is expressed diffusely throughout the GT at all developmental stages, increasing as development progresses. Additionally, there are certain regions of the GT with consistently higher Ar expression, particularly in the urorectal septum and central penile mesenchyme. At GD15 and GD16, when Ar expression is generally lower than at later stages, the urorectal septum is the region with highest Ar expression ( Figures 3A, B , black arrowheads). At GD17 and GD18, the entire cell-dense urorectal septum is expressing Ar ( Figures 3C, D , black arrowheads) but as it grows into the urethral plate, Ar-positive cells are most densely located just ventral to the urethra ( Figures 3E-G , black arrowheads). The central penile mesenchyme is seen from GD17, at which point it has a slightly higher Ar expression than the surrounding mesenchyme ( Figure 3C ). At GD19, the central penile mesenchyme contains localized areas of high Ar expression in the distal part of the GT ( Figure 3E , red arrowhead), which persist through GD20 and GD21 ( Figures 3F, G , red arrowheads).

Esr1 expression is limited to specific areas of the GT during development. At GD15-16, Esr1 expression is very weak, and only localized to small areas, mainly around the urogenital sinus where expression peaks at GD17 ( Figures 3H-J , black arrowheads). Low Esr1 expression is also seen at the ventral side along the urethral plate at GD16 ( Figure 3I , blue arrowhead), which continues from GD17-19 ( Figures 3J-L , blue arrowheads). From GD19, Esr1 is expressed in the urorectal septum, specifically in mesenchymal cells just ventral to the urethra ( Figures 3L-N , black arrowheads). In the central penile mesenchyme, Esr1 is first seen at GD18, although weakly and restricted to the very distal end ( Figure 3K , red arrowhead). At GD19, more of the central penile mesenchyme expresses Esr1 with the highest expression still being at the distal end ( Figure 3L , red arrowhead). At GD20 and GD21, Esr1 is again mainly expressed in the distal tip of the glans central penile mesenchyme, as well as on the dorsal side of the urethra ( Figures 3M, N , red arrowheads). Lastly, when the prepuce starts appearing at GD18, a thin layer of Esr1-positive cells lies beneath the epithelium in both the dorsal and ventral developing prepuce ( Figures 3K-N ).

Having mapped the expression patterns of Ar and Er during normal penis development in rats, we next investigated how the expression was affected by an EDC inducing hypospadias. To have a model of hypospadias, we utilized the potent AR antagonist and pharmaceutical flutamide, which has previously been used as a model chemical to induce hypospadias in rats (9, 28). We first validated the hypospadias model by perinatal (GD7-PD28) exposure of rats, via the dams, to flutamide in doses of either 6 or 18 mg/kg bw/day. Dam, pregnancy, and offspring data, including reproductive parameters in male offspring, are provided in Supplementary Tables 2 - 4 . Genital malformations were visible in the flutamide-exposed male rats as early as PD6 ( Supplementary Table 3 ). At PD28, male fetuses exposed to 6 mg/kg bw/day flutamide (one per litter) had either moderate or severe hypospadias, while 10/11 of the male rats exposed to 18 mg/kg bw/day flutamide had feminized external genitalia with the last one in this group having severe hypospadias ( Table 1 ).

We next explored how the hypospadias phenotype develops prenatally. Due to the feminized phenotype caused by 18 mg/kg bw/day flutamide, we replaced this dose with a lower dose of flutamide (3 mg/kg bw/day). Pregnant rats were thus administered flutamide (0, 3, or 6 mg/kg bw/day) from GD7 until necropsy at either GD17, GD19, or GD21. Dam and pregnancy data for litters necropsied at GD21 are provided in Supplementary Table 5 . In the absence of postnatal data for 3 mg/kg bw/day flutamide, we assessed the adverse reproductive effects of flutamide prenatally by examining the GD21 fetuses.

At GD21, AGD was shorter in male fetuses in both flutamide exposure groups compared to control male fetuses ( Figure 4A , 76.9 and 66.7% of control, respectively, p<0.001), with no difference in body weight ( Supplementary Table 6 ). In the GT of control males, the preputial folds were closing at the ventral midline with a single urethral opening near the distal tip of the GT ( Figures 4B, C ). In flutamide-exposed males, the folds were likewise closing in at the midline but there was an opening in the tissue towards the base of the GT ( Figures 4D, F arrowheads); histologically, it was also clear that the urethral opening was more proximally located ( Figures 4E, G , arrows). This appeared to be caused, at least partly, by underdevelopment of the urorectal septum and fusion of the ventral prepuce. Thus, both doses of flutamide disrupted masculinization in the fetuses, with hypospadias phenotypes presenting already prenatally. In general, the phenotype in the higher flutamide dose group (6 mg/kg bw/day) was more severe than the lower flutamide dose group (3 mg/kg bw/day). The morphological and histological phenotypes observed in flutamide-exposed GTs largely resembled the female GT at GD21 ( Figures 4H, I ). Animations of µCT scans are available in Supplementary Files (see Supplementary Data Sheet 1 ).

To decipher how exposure to anti-androgenic substances may alter the hormone signaling pathways in the developing GT, we investigated gene expression levels of Ar and Esr1 at GD17, 19, and 21 by RT-qPCR, comparing control males to flutamide-exposed males and control females ( Figure 5 ). Exposure to 3 mg/kg bw/day flutamide did not change expression of Ar in the GT, but 6 mg/kg bw/day increased Ar expression at GD21 ( Figure 5A , p=0.04). Moreover, Ar expression was higher in control females at GD19 and GD21 compared to control males ( Figure 5A , p=0.03 and p=0.008, respectively). For Esr1, both doses of flutamide decreased its expression in the GT at GD17 ( Figure 5B , p=0.008, and p=0.04 for 3 and 6 mg/kg bw/day, respectively), while on GD19, the expression of Esr1 was increased in flutamide (6 mg/kg bw/day) males ( Figure 5B , p=0.01). There was no difference in Esr1 expression between flutamide-exposed and control males at GD21 ( Figure 5B ). Esr1 expression was higher in control females at both GD19 and GD21 compared to control males ( Figure 5B , p<0.001, and p=0.002, respectively).

Both Ar and Esr1 expression was, at some stage during development, affected by flutamide exposure as observed by RT-qPCR. We thus examined the spatial expression patterns of Ar and Esr1 in the GT of flutamide-exposed fetuses by RNAscope (summary of changes in Supplementary Figure 1 ). We performed semi-quantitative analyses on areas of the GT to assess any regional changes in expression ( Supplementary Figures 2 , 3 ). Despite no quantitative difference in Ar levels at GD17 or GD19 compared to controls ( Figure 5A ), males exposed to 6 mg/kg bw/day flutamide appeared to have lower Ar expression in the urorectal septum at GD17 and in the central penile mesenchyme at GD19 ( Figures 6C, G , black and red arrowheads, respectively). At GD21, Ar expression in the ventral region of the GT, including the urorectal septum and ventral prepuce, was increased in males in both flutamide groups ( Figures 6J, K , black arrowheads). The higher Ar levels at GD19 and GD21 in females ( Figure 5A ) were likewise mainly due to higher expression in the urorectal septum and ventral prepuce ( Figures 6H, L , black arrowheads), although at GD19 concomitant with lower Ar expression in the distal central clitoral mesenchyme ( Figure 6H , red arrowhead). Moreover, a subtle increase in Ar expression in cells beneath the epithelium in the dorsal prepuce was evident at GD21 both in females and in males exposed to 6 mg/kg bw/day flutamide ( Figures 6K, L , blue arrowheads).

In GD17 GTs, there were no noticeable differences between the four groups in the spatial expression of Esr1 ( Figures 7A-D ), despite lower levels being measured in flutamide-exposed males by RT-qPCR ( Figure 5B ). Esr1 expression was increased in the ventral mesenchyme of both flutamide groups at GD19 ( Figures 7F, G , blue arrowheads) and in the central penile mesenchyme of the 6 mg/kg bw/day flutamide group at GD19 and GD21 ( Figures 7G, K , red arrowheads). Noticeably, flutamide did not induce ectopic expression of Esr1 in new areas of the GT, but instead increased levels in the distinct Esr1-positive areas. In females, Esr1 expression was higher compared to control males at GD19 in the central mesenchyme, ventral mesenchyme, and urorectal septum ( Figure 7H , red, blue, and black arrowheads, respectively), and at GD21 in the urorectal septum only ( Figure 7L , black arrowhead).