Postnatal Cell‐Type‐Specific Localization of EGFR and Its Ligands (EGF, TGF‐α and AREG) in the Rat Testis

ABSTRACT

The epidermal growth factor receptor (EGFR) signalling network and its ligands, including epidermal growth factor (EGF), transforming growth factor‐α (TGF‐α) and amphiregulin (AREG), are involved in the regulation of testicular development and function. However, their postnatal cell‐type‐specific localization patterns in the testis remain incompletely understood. This study investigated the postnatal cell‐type‐specific localization patterns of EGFR, EGF, TGF‐α and AREG in the rat testis using histological and immunohistochemical approaches at four developmental stages. EGFR immunoreactivity was predominantly localized in Sertoli cells, peritubular myoid cells and vascular smooth muscle cells, whereas Leydig cells exhibited weaker immunoreactivity and germ cells lacked staining. In contrast, EGF and AREG exhibited strong and persistent expression in Leydig cells throughout postnatal development. TGF‐α demonstrated stage‐dependent immunoreactivity in Leydig cells and spermatids. Although semiquantitative scores did not reveal significant stage‐dependent differences, distinct cell‐type‐specific localization patterns were observed throughout postnatal development. These findings indicate that EGF family signalling in the postnatal rat testis is primarily mediated through somatic cell populations and support the presence of a paracrine regulatory network involved in the organization of the testicular microenvironment and the regulation of spermatogenesis.

1. Introduction

The testis is a highly specialized organ responsible for spermatogenesis and androgen production, both of which are essential for male fertility (Özer 2007; Ross and Pawlina 2011). Androgen production occurs during two distinct developmental phases mediated by fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are responsible for testosterone synthesis during fetal life, whereas ALCs maintain steroidogenic activity during postnatal development and adulthood (Bhattacharya and Dey 2023; Keer Jiang and Jørgensen 2024; Skakkebaek et al. 2016). Following birth, FLCs gradually regress, while ALCs differentiate from interstitial progenitor cells, resulting in increased androgen production (Li et al. 2018).

Steroidogenesis is regulated by several steroidogenic enzymes, among which cytochrome P450 family 11 subfamily A member 1 (CYP11A1) catalyses the conversion of cholesterol to pregnenolone and represents a key marker of steroidogenic activity (Miller and Auchus 2011; Shima et al. 2015). In contrast to ALCs, which possess the complete enzymatic machinery required for testosterone synthesis, FLCs exhibit limited expression of certain steroidogenic enzymes and partially depend on Sertoli cell support (O'Shaughnessy et al. 2000; Shima et al. 2015). Because CYP11A1 is consistently associated with steroidogenic function, CYP11A1 immunolabelling was used in this study to identify Leydig cell populations during postnatal development.

Testicular development and function require precise spatial and temporal regulation throughout fetal and postnatal life. In addition to endocrine control mediated by follicle‐stimulating hormone, luteinizing hormone and testosterone, locally produced growth factors within the testicular microenvironment are known to contribute to spermatogenesis, steroidogenesis and tissue differentiation (Abd‐Elmaksoud and Sinowatz 2005; Huleihel and Lunenfeld 2004; Skinner 1991).

Among these regulatory systems, EGFR is a transmembrane receptor tyrosine kinase involved in the regulation of cellular proliferation, differentiation and survival (Herbst 2004). EGFR signalling has been extensively investigated in developmental biology and tissue homeostasis (Ceresa and Peterson 2014; Yarden and Sliwkowski 2001). EGFR is activated by several endogenous ligands, including EGF, TGF‐α and AREG, which differ in receptor affinity, tissue distribution and expression patterns. These differences may influence the intensity, duration, and intracellular localization of downstream signalling pathways (Derynck et al. 1984; Jacobs et al. 2009; Roepstorff et al. 2009; Strachan et al. 2001).

Previous studies have established the importance of the EGF/EGFR signalling network in testicular development. However, these investigations have largely focused on specific developmental periods or individual components of the signalling network. Mullaney and Skinner (1992) demonstrated that TGF‐α and EGFR are predominantly expressed during pubertal testicular development and suggested that TGF‐α functions as an autocrine/paracrine regulator of somatic cell growth, while low levels of EGFR gene expression in germ cells indicated potential Sertoli–germ cell communication. Yan et al. (1998) further highlighted the role of EGF in testicular development and spermatogenesis, proposing that EGF produced by Leydig and germ cells may regulate spermatogenesis through autocrine and paracrine mechanisms. Subsequently, Levine et al. (2000) and Cupp and Skinner (2001) demonstrated that TGF‐α and EGFR are expressed during embryonic and perinatal testicular development, display cell‐type‐specific localization, and contribute to testicular growth and differentiation. Although components of the EGFR signalling network have been identified in the testis, information regarding their postnatal, stage‐dependent and cell‐type‐specific expression patterns remains limited. Most previous studies have focused on individual ligands or restricted developmental periods, resulting in an incomplete understanding of EGF family signalling during testicular maturation. Therefore, this study was designed to characterize the postnatal expression patterns and cell‐type‐specific localization of EGFR and its ligands (EGF, TGF‐α and AREG) in the rat testis during distinct developmental stages.

2. Materials and Methods

2.1. Animals and Experimental Design

All experimental procedures were approved by the Ankara University Animal Experiments Local Ethics Committee (Approval no. 2023‐12‐108, June 21, 2023). Male Wistar albino rats were obtained from the Ankara University Experimental Animals Production and Research Laboratory. Animals were maintained under standard laboratory conditions (20°C–24°C; 12 h light/12 h dark cycle) with free access to standard pellet diet and tap water. To evaluate postnatal testicular development, 24 rats were divided into four experimental groups (n = 6/group): neonatal (postnatal day 5; PND5), infantile (PND20), postpubertal (PND50) and adult (PND70). Animals were euthanized under ketamine (80 mg/kg) and xylazine (10 mg/kg) anaesthesia, and testicular tissues were collected for histological and immunohistochemical analyses.

2.2. Histochemical Analysis

Testicular tissues were fixed in Bouin's solution and processed according to routine histological procedures. Following paraplast embedding, 5 μm‐thick sections were obtained and mounted on glass slides. Histochemical evaluation was performed using Crossmon's modified trichrome staining and the periodic acid–Schiff (PAS) method according to established protocols (Bancroft and Gamble 2002; Crossmon 1937).

2.3. Semi‐Thin Section Preparation

For semi‐thin section analysis, tissue samples were fixed in a glutaraldehyde–paraformaldehyde mixture (pH 7.4) according to Karnovsky (1965), post‐fixed in 1% osmium tetroxide for 2 h and treated with 1% uranyl acetate. Samples were dehydrated through graded alcohol series, cleared in propylene oxide and embedded in Araldite M. Semi‐thin sections (1 μm) were stained with toluidine blue for morphological evaluation.

2.4. Immunohistochemistry

Immunohistochemical analyses were performed using the streptavidin–biotin complex (Strept‐ABC) immunoperoxidase method (Bancroft and Gamble 2002). Five‐micrometre paraffin sections mounted on poly‐L‐lysine–coated slides were deparaffinized, rehydrated and rinsed in phosphate‐buffered saline (PBS). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in 70% methanol/PBS for 20–30 min. Antigen retrieval was performed by microwave heating in citrate buffer for 20 min, followed by cooling for an additional 20 min. Non‐specific binding was blocked using 10% normal goat serum (Ultra V Block, Epredia). Sections were incubated overnight at 4°C with primary antibodies against EGFR, EGF, AREG, TGF‐α and CYP11A1 (Table 1). CYP11A1 immunostaining was used as a steroidogenic marker to identify Leydig cell populations during postnatal development. Following PBS washing, sections were incubated with biotin‐conjugated secondary antibodies and horseradish peroxidase–conjugated streptavidin. Immunoreactivity was visualized using 3‐amino‐9‐ethylcarbazole (AEC) as the chromogen, and sections were counterstained with Gill's haematoxylin. Slides were mounted using an aqueous mounting medium. Positive and negative controls were included in all staining procedures. Brain (EGF), ovary (EGFR), liver (AREG), kidney (TGF‐α) and adrenal gland (CYP11A1) tissues served as positive controls. Negative controls were processed by omitting the primary antibody. Sections were examined and photographed using a Leica DM2500 research microscope (Leica Microsystems, Germany).

TABLE 1.

Primary antibodies used in the study.

Primary antibody	Product code	Clone	Dilution	Supplier
Epidermal growth factor receptor (EGFR)	NBP1‐84814	Polyclonal	1:300	Novus biologicals
Epidermal growth factor (EGF)	PA5‐79188	Polyclonal	1:500	Thermo Fisher
Amphiregulin (AREG)	PA5‐102501	Polyclonal	1:100	Thermo Fisher
Transforming growth factor‐α (TGF‐α)	NBP1‐87501	Polyclonal	1:100	Novus biologicals
CYP11A1	NBP2‐92879	Polyclonal	1:100	Novus biologicals

2.5. Evaluation of Immunohistochemical Staining

Immunostained sections were evaluated semiquantitatively by two independent observers. For each animal, three sections and at least five randomly selected microscopic fields per section were examined. Immunoreactivity was scored using a four‐tier scale: 0 (negative), + (weak), ++ (moderate) and +++ (strong). Data were expressed as median (minimum–maximum) values.

2.6. Statistical Analysis

Statistical analyses were performed using SPSS software (version 21.0). Because immunohistochemical staining scores were ordinal in nature, non‐parametric statistical methods were used. Differences among postnatal groups were analysed using the Kruskal–Wallis test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Structural Characteristics of the Testis

3.1.1. Neonatal (PND5)

The neonatal testis contained seminiferous tubules lacking a distinct lumen and composed of gonocytes (prespermatogonial cells) (Figure 1A,B, arrows) and immature Sertoli cells (Figure 1A,B, arrowheads) in both Crossmon's modified trichrome‐stained and toluidine blue–stained semi‐thin sections. Within the interstitial compartment, FLCs (Figure 1A, yellow arrow) and Leydig cells with adult‐type morphology (Figure 1A, red arrow) were observed. FLCs were arranged in clusters surrounded by a basal lamina.

FIGURE 1.

Neonatal rat testis. (A) Seminiferous tubules containing immature Sertoli cells (arrowhead) aligned along the basal lamina and centrally located gonocytes (arrow). In the interstitial compartment, fetal Leydig cells (yellow arrow) and ALC‐like cells (red arrow) are present. Crossmon's modified trichrome staining. Scale bar: 50 μm. (B) Semi‐thin section showing a gonocyte (arrow) and a Sertoli cell nucleus (arrowhead). Toluidine blue staining. Scale bar: 50 μm.

3.1.2. Infantile (PND20)

During the infantile stage, seminiferous tubules exhibited increased structural organization, and lumen formation had begun (Figure 2A, *). Most gonocytes had migrated toward the basal lamina and differentiated into spermatogonia (Figure 2B, arrow). Primary spermatocytes were also present.

FIGURE 2.

Infantile rat testis. (A) Seminiferous tubules showing the onset of lumen formation (*) and the presence of primary spermatocytes (arrowhead). Crossmon's modified trichrome staining. Scale bar: 50 μm. (B) Semi‐thin section showing type A spermatogonia (black arrow) and type B spermatogonia (red arrow) aligned along the basal lamina, as well as Sertoli cell nuclei (arrowhead). Toluidine blue staining. Scale bar: 20 μm.

3.1.3. Postpubertal (PND50)

At the postpubertal stage, seminiferous tubules contained germ cells at different stages of spermatogenesis (Figure 3A,B). The seminiferous epithelium exhibited a multilayered organization composed of type A spermatogonia (Figure 3B, arrowhead), type B spermatogonia (Figure 3B, arrow), primary spermatocytes (Figure 3A, yellow arrow), secondary spermatocytes (Figure 3A, red arrow), spermatids (Figure 3A, *) and spermatozoa. Sertoli cells (Figure 3A, black arrow) were located along the basal lamina and contained prominent nucleoli. Compared with the adult testis, seminiferous tubules showed lower cellular density and fewer elongated spermatids.

FIGURE 3.

Postpubertal rat testis. (A) Seminiferous tubules showing germ cells at different stages of spermatogenesis. Sertoli cells (black arrow), primary spermatocytes (yellow arrow), secondary spermatocytes (red arrow), spermatids (*) and spermatozoa form a multilayered seminiferous epithelium. Crossmon's modified trichrome staining. Scale bar: 50 μm. (B) Semi‐thin section showing type A spermatogonia (arrowhead), type B spermatogonia (black arrow), and a primary spermatocyte (yellow arrow) aligned along the basal lamina. Toluidine blue staining. Scale bar: 50 μm.

3.1.4. Adult (PND70)

Adult testes exhibited fully developed seminiferous tubules with an organized spermatogenic epithelium. All germ cell types, from spermatogonia to mature spermatozoa, were present within the seminiferous epithelium (Figure 4A,B). Seminiferous tubules displayed increased diameter, and the lumina were densely filled with spermatozoa.

FIGURE 4.

Adult rat testis. (A) Seminiferous tubules showing germ cells at different stages of spermatogenesis. Type A spermatogonia (arrow), type B spermatogonia (arrowhead), primary spermatocytes (yellow arrow), secondary spermatocytes (red arrow), spermatids (blue arrow) and spermatozoa form a multilayered seminiferous epithelium. Crossmon's modified trichrome staining. Scale bar: 50 μm. (B) Semi‐thin section showing Sertoli cells (arrows) located along the basal lamina and differentiating germ cells. Toluidine blue staining. Scale bar: 50 μm.

3.1.5. Leydig Cells

Two distinct Leydig cell populations, FLCs and ALCs, were identified during postnatal development. In neonatal and infantile testes, FLCs and ALC‐like cells coexisted within the interstitial compartment (Figure 5A,B). During the postpubertal stage, the number of FLCs decreased, whereas ALCs became the predominant population. In the adult stage, only ALCs were observed.

FIGURE 5.

Morphological and immunohistochemical characteristics of Leydig cells in the neonatal rat testis. (A) Semi‐thin section showing fetal Leydig cells (FLCs) arranged in clusters (black arrow), fibroblast‐like cells surrounding the clusters (yellow arrow) and ALC‐like cells (arrowhead). Toluidine blue staining. Scale bar: 20 μm. (B) Fibroblast‐like cells surrounding FLC clusters (yellow arrow) and ALC‐like cells (arrowhead) within the interstitial compartment. Crossmon's modified trichrome staining. Scale bar: 50 μm. (C) PAS‐positive basal lamina surrounding FLC clusters (arrow). PAS staining. Scale bar. 50 μm. (D) Cytoplasmic immunoreactivity for CYP11A in FLCs (arrow) and ALC‐like cells (arrowhead). Scale bar: 50 μm. (E) Positive control section (adult rat adrenal tissue). Scale bar: 100 μm. (F) Negative control section. Scale bar: 50 μm.

Semi‐thin and histochemical analyses demonstrated that FLCs were arranged in clusters surrounded by fibroblast‐like cells (Figure 5A,B, yellow arrow). PAS staining revealed a positive basal lamina surrounding FLC clusters (Figure 5C, arrow), whereas ALCs showed a more dispersed distribution within the interstitial compartment (Figure 5A, arrowhead). FLCs were still detectable during the infantile (Figure 6A, arrow) and postpubertal stages (Figure 6B, arrow), whereas only ALCs were observed in the adult testes (Figure 6C, arrows).

FIGURE 6.

Distribution of Leydig cells in rat testes at different postnatal developmental stages. (A) Fetal Leydig cells (FLCs) in the interstitial compartment during the infantile stage (arrow). (B) Persistence of FLCs (arrow) in the interstitial compartment during the postpubertal stage. (C) Adult Leydig cells (ALCs) observed in the adult stage (arrows). Crossmon's modified trichrome staining. Scale bar: 50 μm.

3.2. Immunohistochemical Results

To confirm the identity of steroidogenic Leydig cells, CYP11A1 immunostaining was performed. Cytoplasmic immunoreactivity was detected in both FLCs and ALCs (Figure 5D, arrows and arrowheads). Positive control sections (rat adrenal tissue) and negative control sections are shown in Figure 5E,F, respectively.

3.2.1. Epidermal Growth Factor Receptor (EGFR) Expression

Specific immunoreactivity for EGFR was confirmed using rat ovarian tissue as the positive control (Figure 7A) and omission of the primary antibody as the negative control (Figure 7B).

FIGURE 7.

Control sections of EGFR immunohistochemical staining. (A) Positive control: Positive immunoreactivity for EGFR in ovarian tissue. (B) Negative control: No specific staining in testicular tissue treated with PBS instead of the primary antibody. Scale bar: 50 μm.

At the neonatal stage, moderate (++) EGFR immunoreactivity was observed in peritubular myoid cells (Figure 8A, black arrow) and vascular smooth muscle cells (Figure 8A, arrowhead). Fetal Leydig cells lacked immunostaining (Figure 8A, red arrow), whereas ALC‐like cells showed weak‐to‐moderate immunoreactivity (+/++) immunoreactivity.

FIGURE 8.

Immunohistochemical expression of EGFR in the rat testis at different postnatal stages. (A) Neonatal stage: EGFR immunoreactivity in peritubular myoid cells (black arrow) and vascular smooth muscle cells (arrowhead), with no staining in fetal Leydig cells (red arrow). (B) Infantile stage: EGFR immunoreactivity in peritubular myoid cells (green arrow), vascular smooth muscle cells, Sertoli cells (yellow arrow), and Leydig cells (black arrows). (C) Postpubertal stage: EGFR immunoreactivity in peritubular myoid cells, Sertoli cells (yellow arrow) and Leydig cells (black arrows). (D) Adult stage: EGFR immunoreactivity in peritubular myoid cells, vascular smooth muscle cells, Sertoli cells (yellow arrow) and Leydig cells (black arrows). No specific staining is detected in germ cells (spermatogonia, spermatocytes or spermatids). Scale bar: 50 μm.

During the infantile stage, strong (+++) EGFR expression was detected in Sertoli cells (Figure 8B, yellow arrow). Leydig cells maintained weak‐to‐moderate (+/++) immunoreactivity (Figure 8B, black arrows), while moderate (++) staining persisted in peritubular myoid cells (Figure 8B, green arrow) and vascular smooth muscle cells.

In postpubertal and adult testes, strong (+++) EGFR expression in Sertoli cells was maintained (Figure 8C,D, yellow arrows). Moderate immunoreactivity in peritubular myoid cells and vascular smooth muscle cells remained stable throughout postnatal development, whereas Leydig cells continued to exhibit weak‐to‐moderate staining (Figure 8C,D, black arrows). No specific EGFR immunoreactivity was detected in germ cells at any developmental stage.

Semiquantitative analysis demonstrated no statistically significant differences in testicular EGFR expression among postnatal developmental stages (p = 0.805) (Figure 9). The semiquantitative immunoreactivity scores are summarized in Table 2.

FIGURE 9.

Comparison of semiquantitative EGFR expression scores in the testis across postnatal developmental stages (p = 0.805).

TABLE 2.

Semiquantitative assessment of EGFR expression in the rat testis during postnatal development.

Cell type	Neonatal	Infantile	Postpubertal	Adult
Gonocytes	0	ND	ND	ND
Spermatogonia	ND	0	0	0
Spermatocytes/spermatids	ND	0	0	0
Sertoli cells	0	+++	+++	+++
Leydig cells (FLC)	0	0	0	−
Leydig cells (ALC)	+/++	+/++	+/++	+/++
Peritubular myoid cells	++	++	++	++
Vascular smooth muscle cells	++	++	++	++

Note: Immunostaining intensity was evaluated as follows: 0 (no immunoreactivity), + (weak), ++ (moderate), +++ (strong).

Abbreviation: ND, not detected or not present.

3.2.2. Epidermal Growth Factor (EGF) Expression

Specific EGF immunoreactivity was confirmed using rat brain tissue as the positive control (Figure 10A) and omission of the primary antibody as the negative control (Figure 10B). Strong (+++) EGF immunoreactivity was consistently restricted to Leydig cells throughout all postnatal stages. During the neonatal, infantile and postpubertal stages, both FLCs (Figure 11A–C, arrows) and ALCs (Figure 11A–C, arrowheads) exhibited strong immunoreactivity. In adult testes, staining was confined to ALCs (Figure 11D, arrow). No EGF immunoreactivity was detected in Sertoli cells, peritubular myoid cells, vascular smooth muscle cells or germ cells, including spermatogonia, spermatocytes and spermatids.

FIGURE 10.

Control sections for EGF immunohistochemical staining. (A) Positive control: Strong EGF immunoreactivity in brain tissue (choroid plexus). (B) Negative control: No specific staining in testicular tissue treated with phosphate‐buffered saline (PBS) instead of the primary antibody. Scale bar: 50 μm.

FIGURE 11.

Immunohistochemical localization of EGF in the rat testis during postnatal development. (A) Neonatal stage: EGF immunoreactivity in FLCs (arrow) and ALC‐like cells (arrowhead). (B) Infantile stage: EGF immunoreactivity in FLCs (arrow) and ALC‐like cells (arrowhead). (C) Postpubertal stage: Strong EGF immunoreactivity persists in FLCs (arrow) and adult Leydig cells (ALCs) (arrowhead). (D) Adult stage: EGF immunoreactivity restricted to ALCs (arrow). No specific staining is detected in Sertoli cells, peritubular myoid cells, vascular smooth muscle cells or germ cells (spermatogonia, spermatocytes and spermatids). Scale bar: 50 μm.

Semiquantitative analysis revealed no significant differences in EGF expression among postnatal groups (p = 1.000) (Figure 12). The semiquantitative immunoreactivity scores are summarized in Table 3.

FIGURE 12.

Comparison of EGF expression levels in the testis across postnatal developmental stages (p = 1.000).

TABLE 3.

Semiquantitative assessment of EGF expression in the rat testis during postnatal development.

Cell type	Neonatal	Infantile	Postpubertal	Adult
Gonocytes	0	ND	ND	ND
Spermatogonia	ND	0	0	0
Spermatocytes/spermatids	ND	0	0	0
Sertoli cells	0	0	0	0
Leydig cells	+++	+++	+++	+++
Peritubular myoid cells	0	0	0	0
Vascular smooth muscle cells	0	0	0	0

Note: Immunostaining intensity was evaluated as follows: 0 (no immunoreactivity), + (weak), ++ (moderate), +++ (strong).

Abbreviation: ND, not detected or not present.

3.2.3. Amphiregulin (AREG) Expression

AREG immunoreactivity was evaluated using the same immunohistochemical approach, and staining specificity was confirmed using rat liver tissue as the positive control (Figure 13A) and omission of the primary antibody as the negative control (Figure 13B). Strong (+++) AREG immunoreactivity was consistently observed in Leydig cells at all postnatal stages (Figure 14A–D, arrows). In addition, weak (+) immunoreactivity was detected in vascular smooth muscle cells (Figure 14C, arrowhead). No AREG immunoreactivity was observed in Sertoli cells, peritubular myoid cells or germ cells.

FIGURE 13.

Control sections for AREG immunohistochemical staining. (A) Positive control: Strong AREG immunoreactivity in liver tissue. (B) Negative control: No specific staining in testicular tissue treated with PBS instead of the primary antibody. Scale bar: 50 μm.

FIGURE 14.

Immunohistochemical localization of AREG in the rat testis during postnatal development. (A–D). Neonatal (A), infantile (B), postpubertal (C) and adult (D) stages. Positive immunoreactivity is observed in all Leydig cells (arrows) and in vascular smooth muscle cells (C, arrowhead). No immunoreactivity is detected in Sertoli cells, peritubular myoid cells or germ cells (spermatogonia, spermatocytes and spermatids). Scale bar: 50 μm.

Semiquantitative analysis demonstrated no significant differences in AREG expression among postnatal groups (p = 1.000) (Figure 15). The semiquantitative immunoreactivity scores are summarized in Table 4.

FIGURE 15.

Comparison of AREG expression levels in the testis across postnatal developmental stages (p = 1.000).

TABLE 4.

Semiquantitative assessment of AREG expression in the rat testis during postnatal development.

Cell type	Neonatal	Infantile	Postpubertal	Adult
Gonocytes	0	ND	ND	ND
Spermatogonia	ND	0	0	0
Spermatocytes/spermatids	ND	0	0	0
Sertoli cells	0	0	0	0
Leydig cells	+++	+++	+++	+++
Peritubular myoid cells	0	0	0	0
Vascular smooth muscle cells	+	+	+	+

Note: Immunostaining intensity was evaluated as follows: 0 (no immunoreactivity), + (weak), ++ (moderate), +++ (strong).

Abbreviation: ND, not detected or not present.

3.2.4. Transforming Growth Factor‐α (TGF‐α) Expression

Specific TGF‐α immunoreactivity was confirmed using rat kidney tissue as the positive control (Figure 16A) and omission of the primary antibody as the negative control (Figure 16B). At the neonatal stage, weak‐to‐moderate (+/++) TGF‐α immunoreactivity was detected in Leydig cells (Figure 17A, arrow), whereas gonocytes, Sertoli cells, peritubular myoid cells and vascular smooth muscle cells lacked immunoreactivity.

FIGURE 16.

Control sections for TGF‐α immunohistochemical staining. (A) Positive control: Strong TGF‐α immunoreactivity in kidney tissue. (B) Negative control: No specific staining in testicular tissue treated with PBS instead of the primary antibody. Scale bar: 50 μm.

FIGURE 17.

Immunohistochemical staining of TGF‐α in the rat testis during the neonatal and infantile stages. (A) Neonatal stage: TGF‐α immunoreactivity in Leydig cells (arrow). (B) Infantile stage: TGF‐α immunoreactivity in Leydig cells (arrow) and perinuclear staining in primary spermatocytes at the pachytene stage (arrowhead). Scale bar: 50 μm.

During the infantile stage, weak‐to‐moderate (+/++) immunoreactivity persisted in Leydig cells (Figure 17B, arrow). In addition, moderate (++) perinuclear staining was observed in pachytene primary spermatocytes (Figure 17B, arrowhead).

At the postpubertal stage, TGF‐α immunoreactivity exhibited a stage‐dependent distribution pattern in spermatids. Weak (+) staining was observed in early (Steps 1–8) and late (Steps 15–19) spermatids at Stages I–VIII, whereas intermediate spermatids (Steps 9–14) at Stages IX–XIV displayed moderate (++) immunoreactivity (Figure 18). Moderate (++) perinuclear staining was also present in primary spermatocytes (Figure 18, red arrow), while Leydig cells exhibited moderate‐to‐strong (++/+++) immunoreactivity (Figure 18, arrowhead).

FIGURE 18.

Immunohistochemical staining of TGF‐α in the rat testis during the postpubertal stage. Weak immunoreactivity is observed in early (Steps 1–8) and late (Steps 15–19) spermatids at Stages I–VIII, whereas moderate immunoreactivity is detected in intermediate spermatids (Steps 9–14) at Stages IX–XIV. Staining is localized in the cytoplasm of spermatids. Moderate perinuclear immunoreactivity is present in primary spermatocytes (red arrow). Leydig cells exhibit moderate‐to‐strong staining (arrowhead). Scale bar: 50 μm.

In adult testes, moderate (++) perinuclear staining persisted in primary spermatocytes. Early and late spermatids exhibited moderate (++) immunoreactivity, whereas intermediate spermatids showed strong (+++) staining (Figure 19). Leydig cells also demonstrated strong (+++) immunoreactivity (Figure 19, arrowhead).

FIGURE 19.

Immunohistochemical staining of TGF‐α in the rat testis during the adult stage. Positive immunoreactivity is observed in the perinuclear region of primary spermatocytes (red arrow). Moderate staining is detected in early (Steps 1–8) and late (Steps 15–19) spermatids located at Stages I–VIII, whereas strong staining is observed in intermediate spermatids (Steps 9–14) at Stages IX–XIV. Strong immunoreactivity is also present in Leydig cells (arrowhead). M, mitosis; D, Diplotene. Scale bar: 50 μm.

Semiquantitative analysis revealed no significant differences inTGF‐α expression among postnatal groups (p = 0.863) (Figure 20). The semiquantitative immunoreactivity scores are summarized in Table 5.

FIGURE 20.

Comparison of TGF‐α expression in the testis across postnatal developmental stages (p = 0.863).

TABLE 5.

Semiquantitative assessment of TGF‐α expression in the rat testis during postnatal development.

Cell type	Neonatal	Infantile	Postpubertal	Adult
Gonocytes	0	ND	ND	ND
Spermatogonia	ND	0	0	0
Primary spermatocytes (pachytene, perinuclear)	ND	++	++	++
Spermatids (early)	ND	ND	+	++
Spermatids (intermediate)	ND	ND	++	+++
Spermatids (late)	ND	ND	+	++
Sertoli cells	0	0	0	0
Leydig cells	+/++	+/++	++/+++	+++
Peritubular myoid cells	0	0	0	0
Vascular smooth muscle cells	0	0	0	0

Note: Spermatids classified as: Early = Steps 1–8; Intermediate = Steps 9–14; Late = Steps 15–19. Immunostaining intensity was evaluated as follows: 0 (no immunoreactivity), + (weak), ++ (moderate), +++ (strong).

Abbreviation: ND, not detected or not present.

4. Discussion

Growth factors regulate testicular function through coordinated autocrine and paracrine interactions involved in spermatogenesis, steroidogenesis and tissue organization (Abd‐Elmaksoud and Sinowatz 2005; Gnessi et al. 1997; Huleihel and Lunenfeld 2004; Wald 2005). Although EGF family signalling has been extensively investigated in the testis, most previous studies have focused primarily on embryonic or adult stages, and information regarding postnatal cell‐type‐specific and stage‐dependent expression patterns remains limited.

In this study, postnatal testicular development in the Wistar albino rat was evaluated together with the immunohistochemical expression patterns of EGFR and its ligands (EGF, TGF‐α and AREG) across distinct developmental stages, specific cell populations and phases of the seminiferous epithelial cycle. In addition, the postnatal distribution and differentiation characteristics of fetal and adult Leydig cell populations were examined.

Previous studies in different species have demonstrated that the EGF/EGFR system contributes to the regulation of testicular function, although cellular localization patterns vary considerably depending on species‐specific characteristics (Abd‐Elmaksoud and Sinowatz 2005; He et al. 2009; Huleihel and Lunenfeld 2004; Kassab et al. 2007; Radhakrishnan et al. 1992; Tomsig and Turner 2006; Yan et al. 1998).

4.1. EGFR

EGFR is a transmembrane receptor tyrosine kinase involved in the regulation of cellular proliferation, differentiation and intercellular communication (Ceresa and Peterson 2014; Hynes and Lane 2005; Yarden and Sliwkowski 2001). In this study, EGFR immunoreactivity was predominantly localized in somatic components of the testis, particularly Sertoli cells, peritubular myoid cells and vascular smooth muscle cells, whereas germ cells consistently lacked immunoreactivity.

The presence of EGFR in peritubular myoid cells from early postnatal stages supports the view that these cells actively contribute to the organization of the testicular microenvironment in addition to their structural role. Similar findings have been reported in alpaca and Tibetan yak testes, where EGFR expression becomes more prominent after puberty and may participate in seminiferous tubule organization (He et al. 2009; Pan et al. 2014).

Comparative studies demonstrate marked species‐dependent differences in EGFR localization. In the porcine testis, EGFR has been reported in both somatic and germ cells, with more pronounced immunoreactivity in germ cells depending on the seminiferous epithelial cycle (Caussanel et al. 1996). In contrast, bovine testis shows predominantly germ cell localization (Kassab et al. 2007), whereas in the canine testis strong expression has been described in Leydig cells with weaker staining in the seminiferous epithelium (Tamada et al. 2016). In alpaca and Tibetan yak, EGFR expression is limited before puberty but becomes more widespread across several cell types after puberty (He et al. 2009; Pan et al. 2014), and similar distributions have been reported in primate testis (Radhakrishnan and Suarez‐Quian 1992).

The ability of EGFR to be activated by multiple ligands, including EGF, TGF‐α, AREG and HB‐EGF, highlights the complexity of EGFR‐mediated signalling networks in the testis (Miyamoto et al. 2006; Wee and Wang 2017; Yarden and Sliwkowski 2001). In this context, the localization of EGFR predominantly in somatic and contractile cell populations supports its involvement in the organization of the testicular microenvironment and the indirect regulation of spermatogenesis. The strong EGFR immunoreactivity observed in Sertoli cells differs from the weak expression reported in the canine testis (Tamada et al. 2016), but is consistent with findings in the mouse testis (Suárez‐Quian and Niklinski 1990). The apparent nuclear‐like staining pattern observed in Sertoli cells should be interpreted with caution, as the primary antibody used in this study targets a cytoplasmic epitope. This staining pattern may therefore reflect downstream signalling events rather than true nuclear localization. Indeed, EGFR‐mediated signalling pathways are known to generate nuclear responses that regulate gene expression, proliferation and differentiation (Ceresa and Peterson 2014; Hynes and Lane 2005; Yarden and Sliwkowski 2001). However, immunohistochemical localization alone does not directly demonstrate functional activity, and these observations require confirmation by functional studies.

Overall, EGFR exhibits a cell type‐ and developmental stage‐specific distribution pattern in the testis. Its prominent expression in Sertoli and peritubular myoid cells supports its role in regulating the somatic microenvironment necessary for spermatogenesis, whereas the weaker immunoreactivity observed in Leydig cells may indicate a more variable contribution to steroidogenesis across species (Nakazumi et al. 1996). The absence of EGFR expression in germ cells further suggests that EGFR‐mediated signalling primarily acts indirectly through somatic cell populations (Caussanel et al. 1996; He et al. 2009; Kassab et al. 2007; Pan et al. 2014).

4.2. EGF

The involvement of epidermal growth factor (EGF) in testicular function has long been recognized, particularly in relation to meiotic regulation during spermatogenesis. Tsutsumi et al. (1986) demonstrated that reduced circulating EGF levels suppress meiosis, whereas exogenous EGF administration restores meiotic activity. In this study, EGF immunoreactivity was consistently restricted to Leydig cells throughout postnatal development. Both fetal and adult Leydig cells exhibited strong immunoreactivity during the neonatal, infantile and postpubertal periods, whereas staining in adult testes was confined exclusively to adult Leydig cells. No immunoreactivity was detected in Sertoli cells, peritubular myoid cells, vascular smooth muscle cells or germ cells. This distribution suggests that EGF is primarily associated with Leydig cell function and steroidogenic activity. Although EGF expression was not observed in germ cells, its effects on spermatogenesis may occur indirectly through systemic EGF or via paracrine signalling from Leydig cells acting on EGFR‐expressing Sertoli cells.

Comparative studies indicate considerable species‐dependent variation in EGF localization within the testis. In porcine testis, EGF expression has been described mainly in Sertoli cells, with weaker staining in Leydig cells and spermatogonia (Caussanel et al. 1996). This distribution has been interpreted as evidence that EGF may regulate cell proliferation and differentiation within the seminiferous epithelium through paracrine mechanisms. In contrast, bovine testis shows EGF expression mainly in germ cells, varying according to the seminiferous epithelial cycle (Kassab et al. 2007), and in vitro studies have demonstrated that EGF can stimulate spermatogonial mitosis (Wahab‐Wahlgren et al. 2003). Similarly, Radhakrishnan et al. (1992) reported EGF immunopositivity in spermatocytes and spermatids in the mouse testis. Conversely, in the canine testis, EGF is predominantly localized in Leydig cells, with weaker expression in Sertoli cells and the seminiferous epithelium (Tamada et al. 2016). Although these findings clearly demonstrate species‐dependent differences in EGF localization, the biological mechanisms underlying these differences remain unclear. Further studies are needed to elucidate the molecular basis of these interspecies differences.

In the alpaca testis, EGF immunoreactivity is absent during the prepubertal period but becomes evident in multiple cell types, including Leydig, Sertoli, peritubular myoid and germ cells after puberty (He et al. 2009). This pattern suggests that local EGF production may increase with sexual maturation and contribute to spermatogenic regulation. However, the absence of detectable local expression at early stages does not exclude a functional role, as systemic EGF or other EGF family ligands may exert regulatory effects during early development (Tsutsumi et al. 1986; Wong et al. 2000; Xian 2007).

The Leydig cell‐restricted distribution observed in this study differs from findings in alpaca (He et al. 2009) but is partially consistent with observations in Tibetan yak, where EGF expression is confined to Leydig cells before puberty and becomes more widespread after puberty (Pan et al. 2014). In addition, studies in mouse have shown that EGF gene expression parallels testicular maturation and Leydig cell proliferation (Zhang et al. 1997), and in vitro experiments have demonstrated that EGF stimulates steroidogenesis in Leydig cells (Verhoeven and Cailleau 1986). Taken together, these findings indicate that EGF localization in the testis is species‐dependent. While EGF expression has been reported in Sertoli and germ cells in some species, the Leydig cell–restricted pattern observed in this study supports a primary association with steroidogenesis. This interpretation is consistent with Leydig cell‐focused localization reported in dog, mouse and human testes (Nakazumi et al. 1996; Tamada et al. 2016; Zhang et al. 1997).

Overall, the strong and persistent localization of EGF in Leydig cells supports its involvement in steroidogenesis‐related processes. The absence of immunoreactivity in Sertoli and germ cells indicates that its effects are likely mediated indirectly via paracrine signalling from Leydig cells. Consistent with this view, in vitro studies in rodent, porcine and human testes have demonstrated that EGF stimulates steroidogenesis in Leydig cells (Sordoillet et al. 1991; Syed et al. 1991; Verhoeven and Cailleau 1986).

4.3. AREG

Amphiregulin (AREG) is one of the EGF family ligands that regulates intracellular signalling pathways through activation of EGFR (Hynes and Lane 2005; Wee and Wang 2017). In this study, AREG immunoreactivity was strongly localized predominantly in Leydig cells throughout all postnatal stages, whereas only weak staining was observed in vascular smooth muscle cells. Sertoli cells, peritubular myoid cells and germ cells lacked immunoreactivity.

Information regarding AREG localization in the testis remains limited compared with other EGF family ligands. Therefore, the present findings provide additional evidence regarding the cell‐type‐specific distribution of AREG during postnatal testicular development. The strong and persistent Leydig cell localization of AREG throughout postnatal development suggests that this ligand may primarily participate in steroidogenesis‐related processes and contribute to the regulation of the Leydig cell microenvironment through paracrine signalling pathways.

4.4. TGF‐α

TGF‐α is an EGFR ligand that shares overlapping signalling pathways with EGF but may exhibit distinct biological effects and tissue‐specific expression patterns (Derynck 1988; Josso 1990). In this study, TGF‐α expression in Leydig cells increased from the neonatal stage onwards and became more pronounced during the postpubertal and adult stages. In addition, immunoreactivity observed in spermatids suggests that TGF‐α may be associated not only with Leydig cell function but also with germ cell maturation processes in the testis. However, semi‐quantitative analysis revealed no statistically significant differences among the developmental stages examined.

The detection of TGF‐α immunoreactivity in spermatids, together with EGFR expression in somatic cells, indicates that TGF‐α may exert its effects predominantly through paracrine interactions with EGFR‐positive cell populations (Petersen et al. 2001). Accordingly, TGF‐α appears to contribute to both steroidogenic processes and germ cell maturation, as well as to the organization of the seminiferous epithelium.

Previous studies have demonstrated that TGF‐α exhibits variable distribution patterns in the testis depending on species and developmental stage. In the porcine testis, TGF‐α expression has been reported mainly in fetal Leydig cells during the perinatal period and predominantly in Sertoli cells in adulthood (Caussanel et al. 1996). In this study, Leydig cell expression was partially consistent with these findings; however, the absence of immunoreactivity in Sertoli cells and the prominent expression in spermatids indicate notable differences. In the canine testis, TGF‐α localization is largely restricted to Leydig cells (Tamada et al. 2016). Experimental studies have also shown that TGF‐α can stimulate Sertoli cell proliferation (Petersen et al. 2001). In this context, although no TGF‐α expression was detected in Sertoli cells in this study, it remains possible that these cells respond to TGF‐α via receptor‐mediated or indirect paracrine mechanisms.

4.5. Leydig Cell Dynamics

The prominent expression of EGF family members in Leydig cells underscores the importance of evaluating the postnatal developmental dynamics of Leydig cell populations. As key regulators of steroid hormone synthesis, Leydig cells occupy a central position in the functional organization of the testis. Accordingly, their embryological origin and differentiation processes are critical for understanding testicular biology (Haider 2004; Skakkebaek et al. 2016). Whether FLCs completely disappear after birth remains a matter of debate. Some studies suggest that a small FLC population may persist in the adult testis, although with limited contribution to steroidogenesis (Barsoum et al. 2013; Habert et al. 2001; Shima and Morohashi 2017; Zirkin and Papadopoulos 2018). In Sprague–Dawley rats, FLCs have been reported to persist up to postnatal Day 90 (Ariyaratne and Mendis‐Handagama 2000; Kerr and Knell 1988). In this study, FLCs were prominent during the neonatal stage, decreased during the infantile and postpubertal stages, and were not observed in the adult stage. These findings suggest that the FLC population may regress earlier in Wistar albino rats compared to Sprague–Dawley rats, indicating strain‐dependent differences in Leydig cell dynamics.

CYP11A1 immunopositivity was detected in FLCs, ALCs and ALC‐like cells, confirming the steroidogenic capacity of these cell populations (Culty et al. 2015; Shih et al. 2011). The presence of CYP11A1 immunoreactivity in multiple cell populations during early postnatal stages supports the coexistence of distinct steroidogenic populations and suggests that Leydig cell differentiation may begin earlier than previously proposed. This differs from previous reports indicating that FLCs represent the sole steroidogenic population during early postnatal life (Ariyaratne and Mendis‐Handagama 2000), while ALCs are reported to emerge around postnatal Day 10 (Mendis‐Handagama et al. 1987, 1998). In this context, our findings may indicate that ALC‐like cells can be detected at earlier postnatal stages than previously described.

Recent studies indicate that Leydig cell development involves multiple progenitor populations and complex differentiation pathways (Barsoum et al. 2013; Kumar and DeFalco 2018). FLCs have been suggested to originate from diverse cellular sources, including the coelomic epithelium, neural crest cells, Nestin‐positive perivascular cells and steroidogenic factor 1 (SF1)‐positive cells (Bhattacharya and Dey 2023; Kumar and DeFalco 2018). Although the FLC population declines during the postnatal period, a small subpopulation may persist and contribute to ALC development (Bhattacharya and Dey 2023; Shima and Morohashi 2017). In this study, CYP11A1 immunopositivity in both FLCs and ALCs at postnatal Days 20 and 50 supports this possibility; however, these findings do not directly demonstrate lineage relationships between these cell populations. Furthermore, postnatal FLC dynamics appear to vary depending on species and strain. While FLC numbers decline rapidly after postnatal Day 7 in mice (Hazra et al. 2013; Wen et al. 2016), they may persist longer in other species (Habert et al. 2001; Haider 2004).

Consistent with this variability, FLCs in this study were observed up to the postpubertal stage but were not detected in the adult testis. Although previous studies suggest that a small FLC population may persist in adulthood (Bhattacharya and Dey 2023; Shima and Morohashi 2017), no definitive morphological evidence was observed in this study. Nevertheless, CYP11A1 immunopositivity may indicate a potential contribution of FLCs to the ALC population (Kumar and DeFalco 2018; Shima and Morohashi 2017). Overall, these findings suggest that Leydig cell development during the postnatal period is a dynamic process influenced by species‐ and strain‐specific characteristics.

This study has some limitations that should be considered. The distinction between fetal and adult Leydig cell populations was based primarily on established morphological criteria together with CYP11A1 immunoreactivity rather than lineage‐specific molecular markers. Although the semiquantitative immunohistochemical approach was appropriate for the objectives of this descriptive localization study, future quantitative molecular analyses, such as RT‐qPCR or Western blotting, would further strengthen and validate the present findings. Finally, the apparent nuclear‐like EGFR immunoreactivity observed in Sertoli cells should be interpreted with caution because the antibody used recognizes a cytoplasmic epitope. Therefore, additional high‐resolution imaging techniques would be valuable to clarify the precise subcellular localization of EGFR.

5. Conclusion

In conclusion, the present findings reveal that the EGF family signalling network in the postnatal rat testis extends beyond a simple somatic support mechanism, functioning instead as a highly coordinated, bidirectional paracrine cross‐talk system. Crucially, the stage‐specific expression of TGF‐α in germ cells—particularly in primary spermatocytes and Stage IX–XIV intermediate spermatids—despite their lack of EGFR, coupled with the strong localization of EGFR in Sertoli and peritubular myoid cells, provides compelling morphological evidence that developing germ cells may modulate their surrounding somatic microenvironment. Furthermore, the persistent, restricted expression of EGF and AREG in Leydig cells throughout postnatal development underscores their important roles in maintaining continuous steroidogenic function, while the stage‐dependent distribution of fetal and adult Leydig cell populations highlights the dynamic nature of interstitial differentiation. Ultimately, these spatiotemporal mapping results establish a robust morphological framework, supporting the concept that EGFR‐mediated testicular regulation involves not only somatic‐to‐germ signalling but also paracrine communication that warrants further advanced functional investigations.

Funding

This study was financially supported by the Scientific Research Projects Coordination Unit of Ankara University (BAP) (Project No: TDK‐2023‐3069).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.