Title
Author
DOI
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Special Issue
Volume
Issue
Fibroblast distribution and localization in male reproductive organs: a
mouse model study via lineage tracing
Distribución y localización de fibroblastos en los órganos reproductores
masculinos: un estudio en modelo murino mediante trazado de linaje
1Division of Urology, University of Texas McGovern Medical School, Houston, TX 77030, USA
2Division of Urology, Department of Surgery, Linkou Chang Lung Memorial Hospital, 333 Taoyuan, Taiwan
3Department of Andrology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 210008 Nanjing, Jiangsu, China
4Department of Surgery, University of Texas McGovern Medical School, Houston, TX 77030, USA
5Department of Urology, Affiliated Zhongda Hospital of Southeast University, 210009 Nanjing, Jiangsu, China
6Department of Urology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
*Corresponding Author(s):yanna.cao@uth.tmc.edu (Yanna Cao); tung.shu@uth.tmc.edu (Tung Shu)
† These authors contributed equally.
| History | Submitted: 30 May 2025 | Accepted: 06 August 2025 | Published: 30 September 2025 |
| Copyright: | ©2025 The Author(s). Published by MRE Press. |
Background: Fibroblasts are vital for tissue structure, repair, and
fibrosis in male reproductive organs, but tools for investigating their
localization and distribution are limited. This study employed the collagen type
I alpha 2 Cre recombinase estrogen receptor tandem dimer Tomato
(Col1a2CreERtdTomato (Tom)) lineage tracing mouse model, enabling
tamoxifen-inducible, fibroblast-specific labeling through Cre recombination under
the Col1a2 promoter. This system labels collagen-producing fibroblasts
with tdTomato, enabling lineage tracing and visualization of their organization
in the penis, prostate, and testis. The study aimed to investigate fibroblast
distribution and localization in these organs using this model.
Methods: Male Col1a2CreERTom mice (n = 5–6) received
tamoxifen (1 mg/day/mouse, intraperitoneal, for 5 consecutive days) to induce Cre
recombination and label collagen-producing fibroblasts with tdTomato. Wild type
(WT) control mice (n = 3) also received tamoxifen. Two days after the last
injection, penis, prostate, and testis tissues were harvested, sectioned, and
examined through microscopy on tdTomato epifluorescence. Fibroblasts were
quantified as the percentage of tdTomato+ cells relative to total
cells. Results: Quantification revealed distinct fibroblast distribution
patterns among the organs. In the Col1a2CreERTom mouse, tdTomato+
fibroblasts were most abundant in the penis (75.24 ± 1.6%), followed by
the prostate (26.02 ± 1.4%) and testis (13.97 ± 0.9%). Within the
penis, the subtunical region had the highest density (93.42 ± 0.5%). In
the prostate, fibroblasts were mainly within the fibromuscular stroma; in the
testis, they were in the tunica albuginea and interstitial compartments. No
tdTomato+ cells were observed in WT controls. Conclusions: The
number and location of collagen-producing fibroblasts differ among the penis,
prostate, and testis, and vary within penile compartments. These findings reflect
the organ- and region-specific distribution of Col1a2-lineage fibroblasts, as
revealed by tamoxifen-induced tdTomato lineage tracing. This study provides a
useful tool for further investigation of fibroblast function in male reproductive
health.
Resumen
Antecedentes: Los fibroblastos son esenciales para la estructura tisular, la reparación y la fibrosis en los órganos
reproductivos masculinos, pero existen herramientas limitadas para investigar su localización y distribución. Este estudio
empleó el modelo de ratón de trazado de linaje colágeno tipo I alfa 2 Cre recombinasa receptor de estrógeno; tandem
dimer Tomato (Col1a2CreERtdTomato (Tom)), que permite el marcaje específico de fibroblastos inducible por tamoxifeno
mediante recombinación Cre bajo el promotor Col1a2. Este sistema marca fibroblastos productores de colágeno con
tdTomato, permitiendo el trazado de linaje y la visualización de su organización en pene, próstata y testículo. El objetivo
fue investigar la distribución y localización de fibroblastos en estos órganos utilizando este modelo. Métodos: Ratones
machos Col1a2CreERTom (n = 5–6) recibieron tamoxifeno (1 mg/día/ratón, intraperitoneal, durante 5 días consecutivos)
para inducir la recombinación Cre y marcar fibroblastos productores de colágeno con tdTomato. Los ratones control de
tipo silvestre (WT, n = 3) también recibieron tamoxifeno. Dos días después de la última inyección, se recolectaron pene,
próstata y testículo, seccionaron y examinaron mediante microscopía de epifluorescencia para tdTomato. Los fibroblastos
se cuantificaron como el porcentaje de células tdTomato+ respecto al total de células. Resultados: La cuantificación reveló
patrones distintos de distribución fibroblástica entre órganos. En los ratones Col1a2CreERTom, los fibroblastos tdTomato+
fueron más abundantes en el pene (75.24 ± 1.6%), seguidos por la próstata (26.02 ± 1.4%) y el testículo (13.97 ± 0.9%).
En el pene, la región subtunical presentó la mayor densidad (93.42 ± 0.5%). En la próstata, los fibroblastos se localizaron
principalmente en el estroma fibromuscular; en el testículo, en la túnica albugínea y los compartimentos intersticiales. No se
observaron células tdTomato+ en los controles WT. Conclusiones: El número y la localización de fibroblastos productores
de colágeno difieren entre pene, próstata y testículo, y varían dentro de los compartimentos penianos. Estos hallazgos reflejan
la distribución órgano- y región-específica de los fibroblastos de linaje Col1a2, revelada mediante trazado de linaje inducido
por tamoxifeno con tdTomato. Este estudio proporciona una herramienta útil para futuras investigaciones sobre la función
de los fibroblastos en la salud reproductiva masculina.
Palabras Clave:Fibroblastos que expresan colágeno;Pene;Próstata;Testículo;Trazado de linaje
Cite this article
Tung Shu, Danqing Ren, Yu-Hsuan Chien, Baibing Yang, Jiajing Li, Chunhui Liu, Tien C. Ko, Yanna Cao, Run Wang. Fibroblast distribution and localization in male reproductive organs: a mouse model study via lineage tracing. Revista Internacional de Andrología. 2025; 23(3): 34-44. DOI: 10.22514/j.androl.2025.028
1. Introduction
Fibroblasts are important for maintaining the structure and function of tissues in many organs [1, 2]. They produce extracellular matrix proteins to support the tissue structure and repair [3]. In the male reproductive system, fibroblasts also play essential roles in maintaining normal tissue homeostasis and function [4, 5, 6].
Fibroblasts in the penis have been shown to contribute to the formation of fibrotic plaques in the penile tunica albuginea [7]. Under certain conditions, these fibroblasts produce excessive extracellular matrix proteins, such as collagen, leading to fibrosis and tissue stiffening, which results in penile fibrosis known as Peyronie’s disease [8, 9]. Furthermore, recent studies have also shown that fibroblasts are actively involved in regulating erectile function in the mouse penis [4, 10, 11].
Regarding penile erection, nitric oxide (NO) released from the cavernous nerves and penile vascular endothelial cells plays a major role. NO activates guanylate cyclase, raising the levels of cyclic guanosine monophosphate (cGMP) that causes a cascade of protein phosphorylation. As a result, smooth muscle cells relax, allowing blood to fill the cavernous sinuses to induce and maintain an erection [12]. In addition to this well-established mechanism, recent studies have explored the role of fibroblasts in regulating penile erectile function. Fibroblasts make up over half of the cells in the corpus cavernosum, where they regulate the extracellular matrix and interact with smooth muscle cells to maintain the tissue environment [11]. Zhao et al. [10] identified six types of fibroblasts in the human corpus cavernosum, showing their role in supporting penile structure and regulating cell communication, which is essential for erectile function. Guimaraes et al. [4] demonstrated that fibroblasts also regulate blood flow by controlling norepinephrine levels around blood vessels, promoting vasodilation and supporting erections.
In the prostate, fibroblasts are involved not only in the normal structural framework but also play pivotal roles in pathological conditions, such as benign prostatic hyperplasia and the progression of prostate cancer [13, 14]. In benign prostatic hyperplasia, increased fibroblast activity secretes growth factors like fibroblast growth factor-2 (FGF-2) and insulin growth factor-1 (IGF-1), driving epithelial proliferation and structural changes [15]. In prostate cancer, the loss of fibroblast-dependent androgen receptor (AR) activation contributes to castration-resistant cancer, while cancer-associated fibroblasts, activated by tumor elements, play a central role in supporting tumor progression [16, 17].
Similarly, in the testis, fibroblasts contribute to the regulation of tissue architecture and are implicated in fibrotic events leading to male infertility [18]. Testicular fibrosis can arise from prolonged mast cell inflammation, leading to fibroblast activation [19]. These fibroblasts become myofibroblasts, producing collagen and fibronectin, which causes excessive scarring and tissue overgrowth [20].
Our group has recently [21] developed the collagen type I alpha 2 Cre recombinase estrogen receptor tandem dimer Tomato (Col1a2CreERtdTomato (Tom)) mouse model to genetically label collagen-expressing fibroblasts across multiple mouse organs. This model was generated by crossing Col1a2CreER mice, which express tamoxifen-inducible CreER under the control of the fibroblast-specific Col1a2 promoter, with a tdTomato Cre-reporter line. Upon tamoxifen administration, CreER enters the nucleus and activates tdTomato expression in Col1a2-expressing fibroblasts. This enables permanent and specific red fluorescence labeling of fibroblasts, allowing their distribution and localization to be visualized in mouse tissues. In the current study, we adapted this model to examine the distribution and localization of fibroblasts in the penis, prostate, and testis to further study the role of fibroblasts in male reproductive organs.
2. Materials and methods
2.1 Animals
All animal experiments were performed according to the protocols approved by the Animal Welfare Committee of UTHealth at Houston. All mice were housed in a climate-controlled room with an ambient temperature of 23 °C and a 12:12-h light-dark cycle. Animals were fed standard rodent chow and given water ad libitum. Male mice (6–8 weeks old) were used. All mice were on C57BL/6 background. Col1a2CreER mice [22] (B6.Cg-Tg(Col1a2-cre/ERT,-ALPP)7Cpd/J, Stock #029567, Jackson Laboratory, Bar Harbor, ME, USA) were crossbred with reporter mice tdTomato [21] (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Stock #007914, Jackson Laboratory) and produced Col1a2CreERTom. The resulting animals were genotyped following specific instructions from Jackson Laboratory. The wild-type (WT) sibling littermates were used as control.
2.2 Model description
The Col1a2CreER mouse line harbors a tamoxifen-inducible CreER gene under the control of the fibroblast-specific regulatory sequence of the procollagen alpha-2(I) chain (proα2(I)) collagen gene (Col1a2) [22]. To enable lineage tracing, Col1a2CreER mice were crossbred with a Cre-reporter line carrying a floxed STOP cassette upstream of the tdTomato red fluorescent protein gene, generating the Col1a2CreERTom model [21]. In this inducible system, Cre recombinase remains cytoplasmic and inactive until tamoxifen is administered. Upon tamoxifen binding, the CreER fusion protein translocates into the nucleus and excises the STOP cassette, enabling permanent tdTomato expression in Col1a2-expressing fibroblasts (Fig. 1). This system allows for temporal control and permanent labeling of fibroblasts under physiological conditions, thereby enabling spatial visualization and quantification of fibroblast distribution in various tissues using epifluorescence microscopy.
Fig. 1.The Col1a2CreERTom mouse model. The Col1a2CreER mice carry a CreER gene under the control of a fibroblast-specific promoter segment from the proα2(I) collagen gene (Col1a2). These mice were bred with tdTomato reporter mice, generating the Col1a2CreERTom mice. TAM administration induces Cre recombination and the resulting Cre recombinase cleaves the stop codon, leading to tdTomato expression in collagen-expressing fibroblasts. Cre: cre recombinase; ER: estrogen receptor; tdTomato: tandem dimer Tomato; TAM: tamoxifen.
2.3 Induction of Cre recombination and tissue harvesting
Two groups of mice were included. The experimental group consisted of 5–6 Col1a2CreERTom mice, which were used to trace collagen-expressing fibroblasts upon tamoxifen (Serial#: T5648-5G, Lot#: WX22909V, Sigma-Aldrich, St. Louis, MO, USA) induction. The control group consisted of 3 WT littermate mice that received the same tamoxifen treatment but lacked the Cre allele and therefore did not express tdTomato. Only WT mice treated with tamoxifen were used as negative controls in this study, as we have previously validated in other organ systems that Col1a2CreERTom mice without tamoxifen and tdTomato-only mice do not show background tdTomato expression [21]. Therefore, these groups were not repeated here. Col1a2CreERTom mice and the control mice were administered tamoxifen (1 mg/day/mouse, for 5 consecutive days, i.p.) to induce Cre recombination. Two days after the final tamoxifen injection, the mice were euthanized using an overdose of isoflurane (J123028, Dechra, Overland Park, KS, USA), followed by cervical dislocation. The penis, testis, and prostate were then harvested for further analysis. This two-day interval allowed time for CreER nuclear translocation, locus of X-over P1 (LoxP) recombination, and the reporter gene expression to reach detectable levels in fibroblasts, as previously demonstrated [22]. The tissue samples were fixed in 10% formalin and paraffin embedded for morphological studies, or fixed in 4% paraformaldehyde and 10% sucrose, followed by optimal cutting temperature (OCT) block preparation for frozen sectioning [23] for epifluorescence.
2.4 Morphological examination
Paraffin-embedded tissue samples of mouse penis, prostate, and testis were sectioned (5-µm thick) in both cross-sectional and longitudinal planes, while prostatic tissues and testicular tissues were sectioned transversely, and stained with hematoxylin and eosin (H&E).
2.5 Quantification of fibroblasts
Frozen tissue blocks were sectioned at 5 µm using a cryostat (Leica CM1850, Leica Biosystems, Buffalo Grove, IL, USA) for epifluorescence of tdTomato. Penile tissues were sectioned in both cross-sectional and longitudinal planes, while prostatic tissues and testicular tissues were sectioned transversely. Nuclei were counterstained with VECTASHIELD Antifade Mounting Medium with DAPI (DAPI: 4′,6-diamidino-2-phenylindole; Lot#: H-1200, Vector Laboratories, Newark, CA, USA). Epifluorescence images were captured using a Nikon Ti-E inverted fluorescence microscope (Nikon Instruments, Melville, NY, USA) equipped with appropriate filter sets for tdTomato and DAPI. Non-overlapping high-power field images (4–6 images/section) were acquired using a Nikon Tie microscope, and tdTomato+ fibroblasts were quantified using Nikon Elements BR 5.30.05 software (Nikon Instruments, Melville, NY, USA) as previously described [24]. The number of tdTomato+ cells was quantified by identifying cells with colocalization of tdTomato fluorescence and DAPI nuclear staining. The result was expressed as a percentage of tdTomato+ cells relative to the total number of DAPI+ nuclei within each tissue region. tdTomato fluorescence represents fibroblasts that have undergone tamoxifen-induced Cre-mediated recombination in the Col1a2CreERTom mouse model. Thus, all quantified tdTomato+ cells reflect lineage-traced collagen-producing fibroblasts [21].
2.6 Statistical analysis
Data are expressed as mean ± standard error (SEM). Statistical significance was determined using one way Analysis of Variance (ANOVA). p values < 0.05 were considered statistically significant. All statistics were performed using GraphPad Prism version 9 (GraphPad Software, Inc., San Diego, CA, USA).
3. Results
3.1 Collagen-producing tdTomato+ fibroblasts in penis, prostate, and testis were efficiently labeled and tracked
As shown in Fig. 2A, tdTomato+ fibroblasts in the studied organ tissues of the Col1a2CreERTom mouse appeared red. After tamoxifen injection, tdTomato+ fibroblasts constituted 75.24 ± 1.6% of total cells in the penis, 26.02 ± 1.4% in the prostate, and 13.97 ± 0.9% in the testis of the Col1a2CreERTom mouse (Fig. 2B). The distribution of tdTomato+ fibroblasts among these three reproductive organs demonstrated a statistically significant difference (p < 0.05). Specifically, the percentage of tdTomato+ fibroblasts was 49.22% higher in the penis than in the prostate and 61.27% higher in the penis than in the testis. In contrast, as shown in tdTomato epifluorescence images, no tdTomato+ cells were observed in these organs of tamoxifen-injected WT control mice (Fig. 2A). Based on the previous findings in the pancreas in our group [21], tdTomato+ cells were positive for the fibroblast marker desmin [25], but negative for peanut agglutinin (PNA, an acinar epithelial cell marker) and cluster of differentiation 31 (CD31) (an endothelial cell marker), confirming that the labeled cells were fibroblasts. This specificity ensures that the model can be reliably used to trace and study fibroblast populations in the other organs.
Fig. 2.The tdTomato+ fibroblasts in the penis, prostate, and testis of Col1a2CreERTom mouse were efficiently labeled and tracked. (A) Representative images of tdTomato epifluorescence and H&E. White asterisk = tunica albuginea around the testis with tdTomato+ fibroblasts. Scale bar = 100 µm. (B) The amount of tdTomato+ fibroblasts in Col1a2CreERTom mouse penis, prostate, and testis. Data are presented as mean ± SEM. n = 5–6 mice/group. DAPI: 4′,6-diamidino-2-phenylindole; TAM: tamoxifen; tdTom: tandem dimer Tomato; tdTomato+: tdTomato positive; H&E: hematoxylin and eosin; Col1a2CreERTom: collagen type I alpha 2 Cre recombinase estrogen receptor tandem dimer Tomato. p < 0.05.
3.2 Distribution and localization of tdTomato+ fibroblasts in penis
In both cross sections (Fig. 3A) and longitudinal sections (Fig. 3B) of the Col1a2CreERTom mouse penis, tdTomato+ fibroblasts were seen spread throughout the penis, but predominantly distributed around the vascular structures, with the majority primarily surrounding the sinusoids within the corpus cavernosum (Fig. 3A, white asterisk).
The tdTomato+ fibroblasts in the Col1a2CreERTom mouse penis were quantified both overall and at three divided regions as previously studied by Guimaraes et al. [4]: the tunica albuginea, the subtunical, and the trabecular region in the corpora cavernosa. The overall percentage of tdTomato+ fibroblasts was around 75.24 ± 1.6% of total cells (Fig. 3C). A significant difference was observed among the three regions (p < 0.05), with a 39.44% higher tdTomato⁺ fibroblast percentage in the subtunical (93.42 ± 0.5%) compared to the trabecular region (53.98 ± 2.6%), and a 15.09% difference between the subtunical and tunica albuginea (78.33 ± 1.8%) (Fig. 3C). This shows that fibroblasts are not evenly distributed, and their presence may relate to the function of each area.
Fig. 3.Distribution of tdTomato+ fibroblasts in both cross (A) and longitudinal (B) sections of the Col1a2CreERTom mouse penis. (A) Cross sections showing representative images of tdTomato epifluorescence and quantification in three regions of penis: the tunica albuginea (TA), the subtunical (ST) and the trabecular region (TR) in the corpora cavernosa (CC). White asterisk = sinusoids with tdTomato+ fibroblasts. Scale bar = 100 µm. (B) Longitudinal sections of the penis. Scale bar = 100 µm. (C) tdTomato+ fibroblasts quantification in the three regions mentioned in (A). Data are presented as mean ± SEM. n = 6 mice/group. tdTomato+: tdTomato positive; DAPI: 4′,6-diamidino-2-phenylindole. p < 0.05.
3.3 Distribution and localization of tdTomato+ fibroblasts in prostate and testis
In the prostate lobes, tdTomato+ fibroblasts were mostly found in the fibromuscular stroma (Fig. 4A, white asterisk), surrounding the secretory epithelial cells. These fibroblasts were spread evenly across the stromal areas, with a percentage of 26.02 ± 1.4% (Fig. 2).
The testis is covered by a thin, tough fibrous layer called the tunica albuginea. The seminiferous tubules are surrounded by a basal lamina that separates them from the interstitial space, provides structural support, and controls the function of nearby cells [26]. From our observations, the tdTomato+ fibroblasts in testis were mostly located in the tunica albuginea around the testis (Fig. 2A, white asterisk), in the basal lamina surrounding the seminiferous tubules (Fig. 4B, yellow asterisk), and around individual stromal cells located between the seminiferous tubules in the interstitial compartment (Fig. 4B, green asterisk). The tunica albuginea of the testis is thin, compact, and easy to separate from testis, making it hard to quantify tdTomato+ fibroblasts under epifluorescence imaging. Therefore, we could only count them in the seminiferous tubules and stromal areas, which constitute 13.97 ± 0.9% of total cells (Fig. 2).
Fig. 4.The tdTomato+ fibroblasts distribution and localization in Col1a2CreERTom mouse prostate and testis. Representative images of tdTomato epifluorescence and H&E. (A) Prostate. White asterisk = fibromuscular stroma with tdTomato+ fibroblasts. (B) Testis. Yellow asterisk = basal lamina with tdTomato+ fibroblasts. Green asterisk = individual stromal cells with tdTomato+ fibroblasts. Scale bar = 100 µm. TAM: tamoxifen; tdTom: tdTomato; H&E: hematoxylin and eosin; Col1a2CreERTom: collagen type I alpha 2 Cre recombinase estrogen receptor tandem dimer Tomato.
4. Discussion
Studying fibroblasts is challenging due to their heterogeneity and the lack of specific markers. Genetic tools in mice, such as colorimetric reporters and Cre recombinase, have been used to study fibroblasts in organs prone to fibrosis, like the heart, kidney, liver, lung, and skeletal muscle [27]. The Col1a2 gene has been used as a model to study how transcription is regulated, especially in connection to the deposition of the extracellular matrix in normal and fibrotic conditions [28]. Col1a2CreER is a transgenic mouse model where the CreER gene is controlled by the Col1a2 promoter, which targets cells that produce type I collagen. Recently, our group [21] used the Col1a2CreERTom model for lineage tracing of collagen-expressing fibroblasts in both normal pancreas and during acute pancreatitis, demonstrating specificity in labeling pancreatic fibroblasts. We also investigated [21] tdTomato+ fibroblasts across multiple organs in this mouse model and observed a wide variation in their distribution pattern, with significantly higher numbers in the lung, skin, and colon, and much lower levels in the kidney, heart, and liver. A recent study by Guimaraes et al. [4] used the solute carrier family 1 member 3-Cre recombinase estrogen receptor T2; Rosa26-loxP-tdTomato (Slc1a3-CreERT2; Rosa26-tdTomato) mice to mark and trace fibroblasts expressing the Slc1a3 protein, which allowed for tracking fibroblast behavior in the penile tissue.
In the current study, we used the Col1a2CreERTom mouse model to identify and quantify collagen 1a2-producing fibroblasts in mouse reproductive organs—the penis, prostate, and testis. We found that tdTomato+ collagen-expressing fibroblasts are present in these three organs, but their amount and distribution are variable.
Research on penile fibroblasts primarily focuses on their role within the tunica albuginea, the fibrous layer surrounding the corpora cavernosa, as they are involved in Peyronie’s disease [29, 30]. In our study, we observed that tdTomato⁺ fibroblasts are distributed throughout the penis, with the highest density in the subtunical region of the corpora cavernosa, followed by the tunica albuginea, and the lowest density in the trabecular region. Similarly, the study by Guimaraes et al. [4] utilized the Slc1a3-CreERT2; R26R-tdTomato mouse model to label Slc1a3 + tdTomato+ cells, primarily fibroblasts, located mostly in the corpora cavernosa. They found that fibroblasts are the most abundant cell type in the corpora cavernosa, significantly exceeding the numbers of vascular smooth muscle cells and endothelial cells. These fibroblasts are broadly distributed in the subtunical and trabecular areas near the vasculature, as well as in the tunica albuginea [4]. Taken together, our findings and those from Guimaraes et al. [4] demonstrated that, the spatial arrangement of fibroblasts is predominantly in the subtunical region and tunica albuginea, suggesting a potentially distinct role in supporting vasodilation and maintaining structural integrity within the corpora cavernosa. tdTomato+ fibroblasts are concentrated around vascular structures, especially sinusoids in the corpus cavernosum, further implicating a role in modulating blood flow during erection. Recent research on the pathophysiology of erectile dysfunction (ED) has explored the important role of fibroblasts in the corpus cavernosum and how they contribute to the disease through the combination of various techniques, such as, single-cell RNA sequencing (scRNA-seq), cell-cell communication networks, etc.[10, 11, 31, 32]. Previously seen as static cells, fibroblasts are now understood to regulate blood flow, fibrosis, and extracellular matrix changes, reflecting the complex interactions between fibroblasts, smooth muscle cells, and endothelial cells. These findings could provide a starting point for new therapeutic options for ED [4, 10, 11, 31, 32]. The quantification ability of the Col1a2CreERTom mouse model will help in studying the complex functions of fibroblasts and support further research into the mechanisms of ED management.
The prostate, a fibromuscular gland that produces prostatic fluid, relies on fibroblasts for development and cancer progression [33]. Studies have aimed to identify fibroblast populations in normal mouse and human prostates, but the diversity of fibroblasts and the lack of specific markers are challenging [5, 16, 33, 34, 35]. Lineage tracing studies could help clarify fibroblast behavior and their role in prostate health. In this study, we found that tdTomato+ fibroblasts were distributed throughout the fibromuscular stroma of the mouse prostate, comprising 26.02% of the total cell population. To our knowledge, no prior studies have reported the absolute proportion of fibroblasts in the wild-type mouse prostate. Previous histological work [33] estimated fibroblast-to-smooth muscle density ratios in defined regions, but organ-wide quantification of fibroblasts has not been described. As our labeling method does not perturb fibroblast homeostasis, we believe this measurement accurately reflects the endogenous abundance of fibroblasts. This quantitative reference provides a valuable baseline for future investigations of stromal remodeling under pathological conditions, including prostate cancer. Our findings support previous reports indicating that fibroblasts are integral components of the prostate stroma [36], potentially contributing to tissue structure and function in both normal and pathological contexts, such as benign prostatic hyperplasia [15, 37]. Furthermore, given the critical role of fibroblasts in modulating the tumor microenvironment, our findings may have implications for understanding stromal remodeling in prostate cancer. Establishing this fibroblast distribution profile in the normal prostate lays the groundwork for future research exploring how fibroblast abundance and activation states may change during disease progression.
In the mouse testis, we observed that most tdTomato+ fibroblasts are mainly distributed in the tunica albuginea surrounding the testis, within the basal lamina surrounding the tubules, and around individual stromal cells between the seminiferous tubules in the interstitial compartment. We measured the proportion of tdTomato+ fibroblasts only in the areas around the seminiferous tubules and in the interstitial compartment and found that they account for about 13.97% of the total cells in these regions. Their location supports the idea that fibroblasts may help maintain the basal lamina and assist the peritubular myoid cells, which play a key role in spermatogenesis [38]. Recent studies using single-cell technologies reveal how fibroblasts interact with other testicular cells, helping to maintain tissue structure [39]. The Col1a2CreERTom mouse model, with its ability to quantify and locate fibroblasts, could become a useful tool for tracking fibroblast activity in the testis, helping to understand their specific role in the testicular microenvironment. When used together with advanced single-cell methods like scRNA-seq, this model may help mapping of fibroblast functions and provide insights into their broader role in maintaining testicular health.
Furthermore, given that the reproductive organs investigated in this study (penis, prostate, and testis) are androgen-dependent [40, 41], our characterization of fibroblast distribution and localization provides an essential foundation for future hormonal investigations. Specifically, fibroblasts expressing collagen may interact closely with AR signaling pathways, potentially modulating tissue responses to hormonal changes [17, 42, 43]. Our findings thus highlight the importance of exploring how hormonal status and androgen signaling influence fibroblast function and remodeling within these organs, which could be pivotal for understanding reproductive physiology and pathology.
This study presents several novelties. Firstly, expanding upon the work of Guimaraes et al. [4], we further confirmed the distribution and localization of fibroblasts within the mouse penis. We used a distinct lineage tracing mouse model and quantified the percentage of fibroblasts across different functional regions of the penis. This presents a more detailed understanding of spatial distribution for further study of the potential roles of fibroblasts within these regions. Additionally, we extended our investigation to the testis and prostate, examining the distribution of fibroblasts in these organs as well. To our knowledge, this is the first study to specifically investigate fibroblast populations in the testis and prostate of male mice using the lineage tracing. This is an important first step toward understanding how fibroblasts may contribute to the function of these reproductive organs.
Despite these novelties, our study also has limitations. Firstly, we did not employ organ-specific markers in addition to the tdTomato labeled fibroblasts. Secondly, this study is descriptive and lacks functional experiments to directly assess the clinical relevance of our findings.
Our current work specifically addresses fibroblast distribution and homeostasis under physiological conditions, providing essential baseline data for normal tissue maintenance. Importantly, this study did not investigate fibrosis or pathological remodeling, and our findings are not intended to imply any fibrotic process. However, future studies incorporating histological methods such as Masson’s trichrome or Sirius Red staining will be valuable to assess whether fibroblast populations contribute to fibrotic changes under pathological conditions.
In addition, evaluating organ-specific biomarkers and performing correlation analyses with fibroblast distribution will be an important next step. Future investigations involving immunofluorescent co-labeling with functional markers such as AR and endothelial nitric oxide synthase (eNOS), as well as hormonal manipulation models, will be crucial to further elucidate the roles of fibroblasts in androgen-regulated reproductive tissues. These approaches will help clarify the functional roles of fibroblasts in different reproductive organs and provide insights into how fibroblast heterogeneity contributes to tissue-specific physiology and disease.
5. Conclusions
This study employed the Col1a2CreERTom mouse model to identify and quantify collagen-expressing fibroblasts in the penis, testis, and prostate. The distinct distribution patterns of the fibroblasts in these male reproductive organs suggested their possible specific roles in tissue structure and function. For example, the high fibroblast density in the subtunical of the penis may contribute to structural integrity and vascular regulation essential for erectile function, while fibroblasts in the prostate stroma may influence epithelial–stromal interactions relevant to benign prostatic hyperplasia or cancer progression. In the testis, fibroblasts localized to the tunica albuginea and interstitial space may play supportive roles in spermatogenesis and testicular immune regulation.
Looking forward, the Col1a2CreERTom model may serve as a platform to explore how fibroblasts contribute to specific disease processes in male reproductive organs. In this study, tdTomato+ fibroblasts were mainly located around vascular structures in the subtunical and trabecular areas of the penis. These regions are important for blood filling during erection. The presence of fibroblasts in these areas suggests they may regulate vascular tone and extracellular matrix stiffness, thereby influencing erectile function. Previous studies also showed that fibroblasts interact with smooth muscle cells and modulate neurotransmitter levels, such as norepinephrine, around blood vessels. These interactions may be related to blood flow and penile tissue relaxation. Future studies can combine this Col1a2CreERTom model with animal models of ED to investigate fibroblast behavior under disease conditions. This may have relevance to aging-related ED or fibrotic disorders such as Peyronie’s disease, where abnormal fibroblast activity leads to localized penile fibrosis and curvature. Functional experiments are needed to test how fibroblast activation or depletion affects penile structure and erection. Single-cell RNA sequencing or spatial transcriptomics may help identify fibroblast subtypes that are more active in fibrotic or vasoregulatory pathways. These approaches can improve understanding of fibroblast roles in ED and may help find new therapeutic targets.
In the prostate, this model can be applied to test how different fibroblast subtypes contribute to stromal growth in benign prostatic hyperplasia or support tumor progression in prostate cancer, where fibroblast–epithelial interactions are key components of disease pathology. In the testis, further investigations are warranted to elucidate fibroblast roles in supporting the spermatogenic environment or promoting fibrosis related to infertility.
Overall, this study provides essential baseline data on fibroblast distribution and homeostasis under physiological conditions. These findings lay the groundwork for future functional studies aimed at understanding fibroblast dynamics in male reproductive organ health and disease. The Col1a2CreERTom model may also facilitate the discovery of novel fibroblast markers or therapeutic targets for diagnostic and interventional strategies in male reproductive diseases.
Availability of data and materials
The data presented in this study are available on reasonable request from the corresponding author.
Author contributions
TS, DR, BY, JL, CL and YC—conducted relevant experiments. TS, DR, YHC, YC and RW—contributed to the data acquisition; processing; analysis; and interpretation. TS and DR—drafted the manuscript. DR, YHC, YC, TCK and RW—revised and refined the manuscript. The study was conceptualized and designed through the collaborative efforts of all authors. All authors have read and approved the final version of the manuscript.
Ethics approval and consent to participate
All animal experiments were performed according to the protocols (AWC-23-2024) approved by the Animal Welfare Committee of UTHealth at Houston.
Acknowledgment
The authors thank the HistoCore Lab at UTHealth Houston for the tissue process and H&E staining, and Amanda C. Bean from the University of Texas McGovern Medical School for her support with English language editing and stylistic revisions.
Funding
This study is partially supported by the NIH1R21 AA027014-01A1 and Jack H Mayfield M.D. Distinguished Professorship in Surgery (TCK). Additional support was provided by the Weatherhead Fund for Translational Research (TS) and the Cecil M. Crigler, M.D. Chair in Urology Endowment Fund (RW). The above sponsors were not involved in the study design, collection, analysis, or interpretation of data.
Conflict of interest
The authors declare no conflict of interest.
