Moderate Intensity Exercise Training in Combination with L-Carnitine Ameliorates The Varicocele-Suppressed Self-Renewal; Implication on Gdnf/C-ret/Gfrɑ1 Complex Signaling in Rats

Abstract

Background:

For the first time, this study aimed to investigate the potential protective effects of L-carnitine and moderate-intensity exercise training (MIET), both individually and in combination, against varicocele (VCL)-induced impairment of spermatogonial stem cell (SSC) self-renewal.

Materials and Methods:

In this experimental study, a total of 36 male Wistar rats (200 ± 20 g) were divided into the following groups: control, VCL (3-month and 5-month), and treatment groups receiving either Lcarnitine (100 mg/kg/day, orally) or MIET for two months, starting three months after VCL induction. After three months of VCL induction, the condition was confirmed by measuring serum testosterone levels and sperm count. The animals in the treatment groups were then subjected to L-carnitine, MIET, or their combination for an additional two months. The expression of key markers involved in SSC self-renewal, including glial cell line-derived neurotrophic factor (Gdnf), its receptors C-ret and Gfrα1, and target genes Etv5 and Bcl6b, was assessed and compared between groups.

Results:

The VCL-sole groups exhibited significant reductions in serum testosterone, sperm counts, and the numbers of Gdnf⁺, Gfrα1⁺, and C-ret⁺ spermatogonia and spermatocytes compared to controls (P=0.001). Lcarnitine and MIET, both individually and in combination, significantly increased Gdnf, Gfrα1, and C-ret expression at mRNA and protein levels, enhanced the numbers of Gdnf⁺, Gfrα1⁺, C-ret⁺, Etv5⁺, Bcl6b⁺, and Nanog+ cells per seminiferous tubule, and improved SSC repopulation index (RI).

Conclusion:

MIET, particularly in combination with L-carnitine, enhances SSC self-renewal by upregulating the Gdnf/C-ret/Gfrα1 signaling pathway and activating downstream target genes, such as Etv5 and Bcl6b.

Introduction

Spermatogonial stem cells (SSCs) are the most primitive type of spermatogonia, known as type A-single (A-s) spermatogonia, which reside as individual cells on the basement membrane of the seminiferous tubules. When A-s spermatogonia differentiate, they produce type Apaired (A-pr) spermatogonia, which remain connected by an intercellular bridge. Conversely, if A-s spermatogonia stay undifferentiated, they generate two A-s spermatogonia through symmetric self-renewal division without forming an intercellular bridge. Self-renewal is governed by various signalling pathways, such as the glial cell linederived neurotrophic factor (Gdnf) and fibroblast growth factor (Fgf) pathways, which maintain a balance between preserving a reserve of undifferentiated cells and allowing some to differentiate (1, 2).

The Gdnf, a member of the transforming growth factor-β superfamily, was initially identified as a survival factor for midbrain dopaminergic neurons (3). It is now recognized as a key regulator in determining the fate of SSCs. Normally secreted by Sertoli cells in the seminiferous tubules, Gdnf promotes the self-renewal of SSCs and inhibits their differentiation when overexpressed in mouse testes (2). In contrast, mice with one Gdnf-null allele gradually lose undifferentiated spermatogonia, leading to Sertoli cell-only seminiferous tubules (4). These findings underscore Gdnf's essential role in spermatogenesis through paracrine signaling. Undifferentiated spermatogonia express the Gdnf receptor, composed of Gdnf-family receptor α1 (Gfrα1) and c-Ret receptor tyrosine kinase (C-ret). Therefore, the Gdnf/Gfra1/C-ret complex in SSCs initiates the cascade of signaling (Bcl-6b/Etv5) that promotes SSC proliferation and maintenance (5, 6).

Varicocele (VCL) is characterized by abnormal venous dilation and/or tortuosity of the pampiniform plexus in the scrotum (7). While VCLs are typically larger and more common on the left side, up to 50% of affected men have bilateral VCL (8). VCL is reported in 15% of the general male population, in 35% of men with primary infertility, and in up to 80% of men with secondary infertility (7, 9). Several previous scholarly studies have demonstrated various pathophysiological mechanisms by which VCL negatively impacts spermatogenesis. These include apoptosis, disruption of endocrine status (10), inflammation (11, 12), oxidative stress (13), epigenetic disorders (14), and disruption of tissue homeostasis through altered expression of heat shock proteins (15) have been identified as the primary mechanisms by which VCL affects spermatogenesis. Recently, it has been shown that VCL reduces the ability of Sertoli cells to synthesize and secrete Gdnf and disrupts the expression of Gfrα1 and c-Ret, thereby impairing the self-renewal process of SSCs (16).

L-carnitine, a hydrophilic quaternary ammonium cation, can be obtained either from external sources such as poultry, fish, and meat or synthesized endogenously by metabolically active organs like the kidney, brain, liver, and various muscle systems. In cells, L-carnitine is crucial for transporting long-chain fatty acids into mitochondria for energy production via β-oxidation (17). Additionally, it possesses antioxidant properties, protecting cellular components from oxidative damage (18). In the male reproductive system, L-carnitine is highly concentrated in the epididymis, spermatozoa, and testis. Its concentration in epididymal fluid is approximately 2000 times higher than in blood, facilitated by an active transport system in the epididymis (17). Studies have shown that L-carnitine positively affects spermatogenesis and sperm maturation (19). L-carnitine has been shown to aid in the recovery of spermatogenesis post-injury, potentially due to its antiapoptotic properties (20). It also influences the expression of various growth factors, including the enhancement of expression of insulin-like growth factor 1 (Igf-1), and modulates the expression of Fgf and nerve growth factor (Ngf) (21-23). Through these effects, L-carnitine contributes to improved cellular function, enhanced tissue repair, and overall organismal growth.

Moderate-intensity exercise training (MIET) involves physical activity performed at a level that raises the heart rate and breathing, yet still allows for conversation. This form of exercise has been shown to improve cardiovascular health, increase stamina, and support metabolic function. MIET has been shown to have beneficial effects on spermatogenesis by enhancing testicular antioxidant capacity (24, 25), reducing apoptosis (26), improving endocrine status (27-29), and modulating the expression of heat shock proteins (30, 31). Recently, MIET has been shown to ameliorate the Gdnf/Gfrα1/C-ret signaling pathway under diabetic conditions (32).

L-carnitine enhances the beneficial effects of exercise by promoting the transport of long-chain fatty acids into mitochondria, thereby increasing energy production and reducing muscle fatigue (33). It helps in the reduction of exercise-induced muscle soreness and damage by lowering oxidative stress and inflammation (34, 35). Additionally, it facilitates quicker recovery post-exercise by decreasing muscle glycogen depletion and accelerating muscle repair processes (36). Overall, L-carnitine supplementation supports enhanced athletic performance and recovery.

Recent studies have provided important new insights into the molecular mechanisms underlying varicocele-associated infertility and the potential therapeutic interventions targeting SSC self-renewal. For instance, emerging evidence suggests that VCL alters the testicular microenvironment not only through oxidative stress and inflammation but also by disrupting mitochondrial function and metabolic signaling pathways essential for SSC maintenance (37). Furthermore, recent work has highlighted the role of exercise in modulating testicular angiogenesis and reprogramming testicular metabolism, thereby supporting SSC function under pathological conditions (38). Similarly, L-carnitine has been reported to exert protective effects by regulating mitochondrial dynamics, improving redox balance, and modulating growth factor-related signalingin male infertility models (39). By integrating these recent findings, our study aims to build upon the latest research to clarify how L-carnitine and MIET, individually and in combination, influence the Gdnf/Gfrα1/Cret signaling axis and SSC self-renewal in a VCL model.

Given the adverse impact of VCL on SSC self-renewal via the Gdnf/Gfrα1/C-ret signaling pathway, combined with the beneficial effects of MIET on this pathway and the protective role of L-carnitine on testicular tissue and exercise-associated outcomes, this study investigates the individual and combined effects of MIET and L-carnitine on VCL-induced damage. To achieve this, the expression levels of Gdnf, its receptors (<italic>Gfrα1</italic> and <italic>C-ret</italic>), and Etv5, a key initiator of the self-renewal process, were assessed both before and after the administration of L-carnitine and MIET. The duration of eight weeks was chosen because previous studies have shown that significant alterations in SSC populations, testicular architecture, and Gdnf/ Gfrα1/C-ret signaling can be observed within this timeframe in rodent VCL models. This period is sufficient to capture the effects of L-carnitine and MIET on SSC selfrenewal and testicular recovery. Moreover, eight weeks corresponds approximately to a full spermatogenic cycle in rats, ensuring that the observed outcomes reflect comprehensive changes in spermatogenesis. The findings aim to elucidate the effects of MIET and L-carnitine on VCL -induced impairments in SSC self-renewal and determine whether L-carnitine enhances the therapeutic benefits of MIET.

Materials and Methods

Experimental design and grouping

In this experimental study, a total of 36 male Wistar rats (200 ± 20 g) were procured from the Laboratory Animal Resource Center at Urmia University (ARCUU). Prior to the experiment, the rats underwent a one-week acclima tization period under a 12-hour light/dark cycle and at a controlled temperature of 24 ± 1ºC, during which they received a standard diet and had access to filtered tap water. Following adaptation, the rats were randomly divided into sedentary control group (con-sham, n=6), VCL-sole (euthanized following 3 and 5 months, n=12), L-carnitinesole-treated (VCL+L-carnitine, 100 mg/kg, daily, for 2 months, orally, n=6) (20), MIET-induced (VCL+Exer, for 2 months, n=6), and L-carnitine+MIET-received VCLinduced groups (n=6).

All procedures adhered to the ethical guidelines set by the Veterinary Ethics committee of Urmia University (IRUU-AEC-3/75). Additionally, The study was conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH, 8th edition) and the European Union Directive 2010/63/ EU for the protection of animals used for scientific purposes. Efforts were made to minimize animal suffering and to reduce the number of animals used, in line with the 3Rs principle (Replacement, Reduction, and Refinement). Accordingly, six out of twelve rats from VCl-induced group were euthanized after 3 months to confirm appropriate VCL induction in the rats (VCL-3). The remained rats were continued solely (VCL-5) for an additional 2 months. The treated groups continued for an additional 2 months to investigate the effects of L-carnitine and MIET in sole and combination on VCL-related damage. Refer to the schematic diagram illustrating group allocation and methodology (Fig .1A, B). Entry criteria were: adult male wistar rats aged 8-10 weeks, weighing 200-250 g, with no prior history of systemic illness or reproductive disorders, were included in the study. Only rats exhibiting normal baseline testicular morphology and sperm parameters were selected for inclusion.

Fig.1.

Comprehensive overview of the experimental design, interventions, testicular morphology, serum testosterone levels, and total sperm count in varicocele-induced rats. A. Schematic view of the animals and grouping, B. Details for the MIET program, C. Gross photomicrographs of the left testicles from control and 3 months VCL-induced groups, q Mean changes in the serum testosterone of 3 months VCL-induced rats in comparison to the control group, E. Mean changes in the total sperm count. All data are presented as mean ± SD, and different stars represent significant differences between groups. MIET; Moderate intensity exercise training, VCL; Varicocele, and ****; P≤0.0001.

Exit criteria: Rats exhibiting severe clinical symptoms, excessive weight loss (>15% of initial body weight), or complications from surgical procedures (e.g., postoperative infection or tissue necrosis) were excluded from the study. Additionally, animals that did not survive the full experimental duration or exhibited abnormal behaviours impacting feeding or physical activity were excluded from data analysis.

Varicocele induction

As described previously (10), experimental left-sided VCL was induced via ligation of the left renal vein. Briefly, after administering anesthesia with 5% ketamine (40 mg/kg, VOLZA, Netherlands) and 2% xylazine (5 mg/kg, VOLZA, Netherlands), the left renal vein was ligated medial to its junction with the adrenal and spermatic veins. Subsequently, the anastomotic branches between the left testicular vein and the left common iliac vein were also ligated. The sedentary control group underwent a simple laparotomy without any ligation.

Maximum velocity test and moderate-intensity exercise training protocols

Following a one-week adaptation period for treadmill running (Danesh Yakhte, Iran, running at an intensity of 15 m/minutes, 5-20 minutes), the maximum velocity test (Vmax) was conducted. The Vmax was determined by a 5-minutes warm-up (at a speed of 10-15 m/minutes), followed by a stepwise increase in speed (5 m/minutes every 3 minutes) until the rats were unable or unwilling to continue, as described in previous literature (40).

After establishing the Vmax, MIET was performed at 60-75% Vmax for 45 minutes. The warm-up and cooldown stages were set at 40-45% Vmax and 35-40% Vmax, respectively. MIET were executed 5 days per week over a 60-day period, following recommendations from previous studies (Fig .1A, B, 40).

Euthanasia and tissue sampling

The rats were euthanized 48 hours after the final exercise session via intraperitoneal administration of an overdose of Xylazine (>20 mg/kg, Alfasan, Woerden, Netherlands) and Ketamine hydrochloride (>100 mg/kg, Alfasan, Woerden, Netherlands). Testicular tissue was carefully dissected, with one-half of the left testes fixed in Bouin’s solution for histological analysis. The remaining testicular tissue was snap-frozen and stored at -80°C for subsequent molecular and biochemical analyses.

Immunohistochemical staining

To evaluate the expression of Gdnf, Gfrα1, and C-ret in testicular tissue, and to quantify SSCs using Nanog (a specific SSC marker), paraffin-embedded cross-sections (5-6 µm thick) were prepared. The distribution and localization of Gdnf⁺, Gfrα1⁺, C-ret⁺, and Nanog⁺ cells within the seminiferous tubules were analyzed and compared across different experimental groups. The immunohistochemical (IHC) staining procedure involved several steps: sections were initially heated at 56°C for 25 minutes, then de-paraffinized in xylene (twice), and rehydrated through graded alcohol solutions (5 minutes each). Antigen retrieval was performed in 10 mM sodium citrate buffer (pH=7.2), followed by blocking endogenous peroxidases with 1.5% hydrogen peroxide in 1x phosphate-buffered saline (PBS) for 20 minutes at room temperature. Sections were then incubated in a SuperBlock solution (SCYTEK Co, AA025, Utah, USA, LOT:43961) for 10 minutes. Primary antibodies against Gdnf (1:300, Abcam, Cat N: ab28956), Gfrα1 (1:500, Elabsciences, Cat N: E-AB-67652), C-ret (1:200, Santa Cruz, Cat N: sc-365943), and Nanog (1:500, Elabsciences, Cat N: E-AB-93110) were applied overnight at 4°C. The target proteins were visualized using 3,3′-diaminobenzidine (DAB) chromogen solution (Sigma, St. Louis, MO) for 5 min, followed by hematoxylin counterstaining for 10 seconds (16). The negative controls were included by replacing the primary antibody with PBS or with nonimmune serum to verify the absence of nonspecific background staining. Positive controls known to express the target proteins (e.g., testicular sections with confirmed expression of Gdnf pathway proteins) were also processed in parallel to validate staining specificity. To ensure reproducibility, all staining was performed using the same optimized protocol, including identical antigen retrieval, blocking, antibody dilutions, incubation times, and detection systems. Furthermore, sections from all experimental groups were stained simultaneously. Two independent, blinded observers assessed staining intensity and distribution to minimize subjectivity and inter-observer variability.

Western blot analysis

Western blot analysis was performed as previously described by Rashtbari et al. (16). Testicular tissues were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, Germany, S8820) to extract proteins. Protein concentration was determined using the Lowry method. Protein samples were then diluted in loading buffer, heated at 95°C for 5 minutes, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 120V. Subsequently, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane at 100V for 1-2 hour. The membranes were blocked overnight with a 5% non-fat milk solution. Following blocking, the membranes were washed three times with Tris-buffered saline (pH=7.2) containing 0.1% Tween 20, with each wash lasting 15 minutes. The membranes were then incubated for 2 hours at 4°C with primary antibodies against Gdnf (Abcam, Cat N: ab28956), Gfra1 (Elabsciences, USA, Cat N: E-AB-67652), C-ret (Santa Cruz, USA, Cat N: sc-365943), Etv5 (Elabsciences, USA, Cat N: E-AB-91955), Bcl-6b (Boster, USA, Cat N: A10281), and β-actin (Elabsciences, USA, Cat N: E-AB-40517). After washing to remove unbound antibodies, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, followed by an additional wash. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Scientific, USA), and band intensities were quantified using an Arash Pishro Teb (ATP) enhanced laser densitometer.

mRNA extraction, cDNA synthesis, and quantitative real-time polymerase chain reaction

mRNA was extracted from tissue samples using the TRIZOL reagent. The concentration and purity of mRNA were evaluated with a Nanodrop spectrophotometer, measuring absorbance at 260 nm and a 260/280 ratio between 1.8 and 2.0. For cDNA synthesis, 1 µg of total mRNA was used in a 20 µl reaction mixture (Pars Toos, Iran). Real-time polymerase chain reaction (PCR) was conducted in triplicate, with each reaction using 0.5 µl of cDNA template (5-10 ng) mixed with 10 µl SYBR GREEN master mix (High ROX, Novaran Teb-Beinolmelal, Iran), 0.6 µl of each forward and reverse primer, and distilled water to a total volume of 20 µl. The PCR protocol included an initial denaturation at 95°C for 600 seconds, followed by 45 cycles of 95°C for 20 seconds, 72°C for 60 seconds, and 72°C for 600 seconds. Expression levels were assessed in triplicate, normalized to β-Actin using the threshold cycle (CT) values, and fold changes were calculated using the formula: 2^{-(Ct target − Ct ϐ-actin)}. Primer sequences used are detailed as follow:

Gdnf-F: 5'-ATGAAGTTATGGGATGTCGTGGCT-3'

R: 5'-GGGTCAGATACATCCACACCG-3'

C-ret-F: 5'-GATGGCACTAGCACTGGGTT-3'

R: 5'-GATGTGCACAAAGTGGTCGG-3'

Etv5-F: 5'-TGTGACCCGGATGCACTTTTCT-3'

R: 5'-TCCATGTCCAGTAGGTAAGCAG-3'

Bcl-6b-F: 5'-AACCCCCAAGACCCCAAC-3'

R: 5'-CTGGACTTGGGGAACCTTG-3'

Gfra1-F: 5'-GCACAGCTACGGGATGCTCTTCTG-3'

R: 5'-GTAGTTGGGAGTCATGACTGTGCCATC-3'

β-actin-F: 5'-GGAGATTACTGCCCTGGCTCCTA-3'

R: 5'-GACTCATCGTACTCCTGCTTGCTG-3'

Statistical and image analyses

The Kolmogorov–Smirnov and Levene’s tests were used to assess the normality and homogeneity of variances of the data, respectively. Quantitative histological, biochemical, and molecular parameters were analyzed using one-way ANOVA, followed by Tukey’s multiple comparison posthoc test where appropriate. Data analyses were analyzed using graphpad prism software (version 11.00, California, USA). A P value of 0.05 was considered significant for all statistical tests, and data were reported as mean ± SD. Graphs were created using GraphPad Prism software (Version 11.00, California, USA). Photomicrographs were captured using a 20-megapixel camera (Canadalab, Canada; Model XTLB200) and analyzed with Image-Pro Insight software (version 9.0; Media Cybernetics, USA). For this analysis, photomicrographs from three randomly selected areas of the cross-section were obtained. The mean pixel -based intensities of the brown-stained reactions, indicative of target proteins, were measured within a 3000 μm×3000 μm tissue area and compared between groups.

Results

The samples after 3 months exhibited a successful varicocele induction

The testicular tissues were dissected, and their crossmonths post-surgery to confirm successful VCL induction. The VCL-induced group displayed prominent varicosities in the peripheral pampiniform (spiral) vessels, whereas the control group exhibited normal vascular morphology. Additionally, serum testosterone levels, a key marker for experimental VCL, were measured. The VCL -induced rats showed a significantly lower serum testosterone level (P=0.001) compared to the control animals. Finally, sperm count analysis revealed a significant reduction (P=0.001) in total sperm count in the VCL-induced rat’s relative to the control group (Fig .1C-E).

Moderate-intensity exercise training and L-carnitine increased Gdnf expression

The VCL-only groups (3- and 5-month time points) exhibited a significant reduction in both Gdnf mRNA (P=0.001) and protein levels (P=0.001) compared with the control-sham group (Fig .2A, B). In contrast, L-carnitine treatment (VCL+L-carnitine group) significantly increased Gdnf mRNA (P=0.001) and protein expression (P=0.001) after 5 months. The MIET (in VCL+Exer group) led to a significant increase in the Gdnf mRNA level (P=0.001), but did not significantly increase the Gdnf protein content compared to the VCL-sole group (P=0.140) after 5 months. The VCL+L+Exer group exhibited a remarkable increase (P=0.001) in the mean Gdnf mRNA and protein levels compared to the VCL-sole group.

Fig.2.

Gdnf expression changes in different experimental groups at transcriptional, translational, and histological levels. A. Mean changes in the mRNA level of Gdnf in different groups, B. Western blot analysis for Gdnf protein content in different groups, C. Immunohistochemistry staining of the Gdnf in cross-sections from different groups. Decreased Gdnf+ spermatogonia (arrowhead), spermatocytes (arrows), and Sertoli cells (thick arrow) in the VCL induced groups, which is ameliorated in the L-carnitine and moderate-intensity training exercise (Exer) groups (scal bar: 80 µm), D. Mean changes in the Gdnf+ spermatogonia, E. Mean changes in the Gdnf+ spermatocytes, F. Mean changes in the Gdnf+ Sertoli cells, G. Mean changes in the pixel-based inten sities for brown reactions representing Gdnf in a 3000 μm×3000 μm area of tissue in different groups. All data are presented as mean ± SD, and different stars represent significant differences between groups (scale bar: 80 µm). Con-sham; Control-sham, VCL-3; Varicocele-induced after 3 months, VCL-5; Var icocele-induced after 5 months, L; L-carnitine, Exer; Moderate-intensity exercise training, ns; P>0.05, *; P≤0.05, **; P≤0.01, ***; P≤0.001, and ****; P≤0.0001.

To investigate the effects of VCL, L-carnitine, and MIET on Gdnf expression in various cell types, immunohistochemistry (IHC) staining was performed. Observations indicated a significant reduction in the mean numbers of Gdnf+ spermatogonia, spermatocytes, and Sertoli cells in the VCL-only groups (at 3 and 5 months) compared to the con-sham rats (P=0.001). This reduction developed in a time-dependent manner in the VCL-sole induced groups (P=0.022). Conversely, L-carnitine (in VCL+L-carnitine group) and MIET (in VCL+Exer group) treatments increased the mean numbers of Gdnf+ spermatogonia (P=0.001, P=0.029), spermatocytes (P=0.001, P=0.028), and sertoli cells (P=0.001, P=0.007) per seminiferous tubule compared to the VCL-sole group. Finally, the VCL+L-carnitine+MIET group demonstrated a significant increase in the mean numbers of Gdnf+ spermatogonia (P=0.001), spermatocytes (P=0.001), and sertoli cells (P=0.001) compared to the VCL-sole rats (Fig .2C-F).

To minimize subjective bias in microscopic evaluation, digital image analysis was applied to quantify the brown DAB-stained signals representing Gdnf expression. The VCL-only group showed a marked decrease in mean pixel-based intensity (measured within a 3000 µm × 3000 µm tissue area) compared to controls (P=0.001). In contrast, L-carnitine (P=0.001) and MIET (P=0.002), either individually or in combination (P=0.001), significantly enhanced Gdnf signal intensity relative to the VCL-only group (Fig .2G).

MIET and L-carnitine, in a simultaneous form of consideration, upregulated the Gfrα1 expression

The VCL-only groups (3 and 5 months) demonstrated a significant reduction (P=0.001) in both mRNA and protein expression levels of Gfrα1 compared with the consham group (Fig .3A, B). Additionally, the mRNA level of Gfrα1 further decreased after 5 months compared to the 3-month VCL-induced rats (P=0.001). However, there was no significant difference in Gfrα1 protein levels between the 3 and 5-month VCL-sole induced groups (P=0.075). In contrast, L-carnitine (in the VCL+L group) significantly increased the mean mRNA (P=0.001) and protein (P=0.001) levels of Gfrα1 compared to the VCL -sole (5-month) rats. The MIET (in the VCL+Exer group) significantly increased the Gfrα1 mRNA level (P=0.001) but did not significantly increase (P=0.069) the protein content when compared to the VCL-only (5 months) group. The L-carnitine and MIET (in VCL+L+Exer) could increase (P=0.001) the Gfrα1 mRNA and protein levels compared to the VCL-sole (5 months) group.

Fig.3.

Gfrα1 expression changes in different experimental groups at transcriptional, translational, and histological levels. A. Mean changes in the mRNA level of Gfrα1 in different groups, B. Western blot analysis for Gfrα1 protein content in different groups, C. Immunohistochemistry staining of the Gfrα1 in cross-sections from different groups. Decreased Gfrα1⁺ spermatogonia (arrowhead) and spermatocytes (arrows) in the VCL-induced groups, which is ameliorated in the L and Exer groups, D. Mean changes in the Gfrα1+ spermatogonia, E. Mean changes in the Gfrα1⁺ spermatocytes, and F. Mean changes in the pixel-based intensities for brown reactions representing Gfrα1 in a 3000 μm×3000 μm area of tissue in different groups. All data are presented as mean ± SD, and different stars represent significant differences between groups (scale bars: 80 µm). Con-sham; Control-sham, VCL-3; Varicocele-induced after 3 months, VCL-5; Varicocele-induced after 5 months, L; L-carnitine, Exer; Moderate-intensity exercise training, ns; P>0.05, *; P≤0.05, **; P≤0.01, ***; P≤0.001, and ****; P≤0.0001.

Similar to Gdnf expression, the mean distributions of Gfrα1+ cells per seminiferous tubule were analyzed and compared between groups. The VCL-sole groups (3 and 5 months) exhibited a significant reduction (P=0.001) in the mean numbers of Gfrα1+ spermatogonia and spermatocyte cells compared to the Con-sham rats (Fig .3CE). There were no significant differences in the mean numbers of Gfrα1+ spermatogonia (P=0.919) and spermatocytes (P=0.579) cells between the 3 and 5 months VCL-sole groups. L-carnitine (P=0.002), but not MIET (P=0.050), significantly increased the mean numbers of Gfrα1+ spermatogonia cells compared to the VCL-sole group. Neither L-carnitine (in the VCL+L group) nor MIET (in the VCL+Exer group) had a significant effect on the mean distributions of Gfrα1+ spermatocyte cells (P=0.570, P=0.336) compared to the VCL-sole (5 months) group. Conversely, the combined treatment of L-carnitine and MIET (VCL+L+Exer group) resulted in a significant increase in the mean number of Gfrα1⁺ spermatogonia and spermatocyte cells compared with the VCL-only group.

Software-based image analysis revealed that VCL significantly decreased the mean pixel intensity (within a 3000 μm×3000 μm area of tissue) of brown-stained reactions compared to the control group (P=0.001). In contrast, both L-carnitine and MIET, whether administered individually or simultaneously (P=0.001), increased the mean intensity value compared with the VCL-only group. This finding suggests enhanced intracellular expression of Gfrα1, rather than an increase in cell number (Fig .3F).

L-carnitine could increase the C-ret expression and MIET could boost the L-carnitine-induced impact

The VCL-only groups (3 and 5 months) exhibited a significant reduction in the mean mRNA and protein levels of C-ret compared with the Con-sham rats (P=0.001). There was no significant difference (P=0.156) in C-ret mRNA expression between the 3- and 5-month VCL-only groups. However, the mean C-ret protein content was significantly decreased (P=0.013) after 5 months compared with the 3-month VCL-only rats (Fig .4A, B). L-carnitine treatment (in the VCL+L group) significantly increased the mean mRNA (P=0.006) and protein (P=0.001) levels of C-ret compared to the VCL-sole (5 months) rats. Conversely, the rats in the VCL+Exer group (received MIET) did not exhibit any significant changes in the mean mRNA (P=0.889) and protein (P=0.136) levels of C-ret when compared to the VCL-sole group (5 months). The combined administration of L-carnitine and MIET (VCL+L+Exer group) significantly upregulated both Cret mRNA and protein levels (P=0.001) compared with the 5-month VCL-only group.

Fig.4.

C-ret expression changes in different experimental groups at transcriptional, translational, and histological levels. A. Mean changes in the mRNA level of C-ret in different groups, B. Western blot analysis for C-ret protein content in different groups, C. Immunohistochemistry staining of the C-ret in cross-sections from different groups. Decreased C-ret+ spermatogonia (arrowhead) and spermatocytes (arrows) in the Varicocele (VCL)-induced groups, which is ameliorated in the L and Exer groups, D. Mean changes in the C-ret⁺ spermatogonia, E. Mean changes in the C-ret⁺ spermatocytes, and F. Mean changes in the pixel-based intensities for brown reactions representing C-ret in a 3000 μm×3000 μm area of tissue in different groups. All data are presented as mean ± SD, and different stars represent significant differences between groups (scale bars: 80 µm). Con-sham; Control-sham, VCL-3; Varicocele -induced after 3 months, VCL-5; Varicocele-induced after 5 months, L; L-carnitine, and Exer; Moderate-intensity exercise training, ns; P>0.05, *; P≤0.05, **; P≤0.01, ***; P≤0.001, and ****; P≤0.0001.

The mean numbers of C-ret+ cells per seminiferous tubule were analyzed and compared between groups (Fig .4C-E). The VCL-sole groups (3 and 5 months) exhibited a significant reduction (P=0.001) in the mean numbers of C-ret⁺ spermatogonia and spermatocyte cells compared to the Con-sham rats. However, L-carnitine and MIET, both individually and in combination, significantly increased (P=0.001) the mean distributions of C-ret⁺ spermatogonia and spermatocytes per seminiferous tubule compared to the VCL-sole (5 months) rats.

The software-based image analysis revealed similar findings, showing a significant reduction (P=0.001) in the mean pixel intensity of brown-stained reactions in the VCL-only groups (3 and 5 months), and a significant increase (P=0.001) following L-carnitine and MIET treatment, both individually and in combination (Fig .4F).

L-carnitine could increase the Etv5 and Bcl-6b expression and MIET could boost the L-carnitine-induced impact

The VCL-sole groups (3 and 5 months) exhibited a significant reduction in the mean mRNA and protein levels of Etv5 and Bcl-6b compared to the con-sham rats (P=0.001). Etv5 mRNA was significantly decreased in the 5-month VCL-sole group compared to the 3-month VCL rats (P=0.002). No significant differences were observed in Etv5 protein levels (P=0.330) between the 3 and 5 months VCL-sole groups (Fig .5A-D).

Fig.5.

Etv5 and Bcl-6b expression changes in different experimental groups at transcriptional, and translational levels. A, B. Mean changes in the mRNA level, C. Photomicrograph of western blot bands, D. Mean changes at protein level. All data are presented as mean ± SD, and different stars represent significant differences between groups. Con-sham; Control-sham, VCL-3; Varicocele-induced after 3 months, VCL-5; Varicocele-induced after 5 months, L; L-carnitine, and Exer; Moderate-intensity exercise training, ns; P>0.05, *; P≤0.05, **; P≤0.01, ***; P≤0.001, and ****; P≤0.0001.

L-carnitine (in the VCL+L group) significantly increased Etv5 mRNA (P=0.001) and protein (P=0.004) levels compared to the VCL-sole group. Additionally, it significantly increased Bcl-6b mRNA (P=0.001) and protein (P=0.007) levels compared to the VCL-sole group (5 months). MIET (in the VCL+Exer group) significantly increased Etv5 and Bcl-6b mRNA levels (P=0.001) compared to the VCL-sole rats. However, no statistically significant increase was observed in Etv5 (P=0.104) and Bcl-6b (P=0.273) protein levels compared to the VCL-sole (5 months) group. Finally, the combined administration of L-carnitine and MIET (VCL+L+Exer group) resulted in a significant upregulation (P=0.001) of both Etv5 and Bcl-6b mRNA and protein -levels compared with the 5-month VCL-only group.

L-carnetin and MIET increased repopulation index by preserving SSCs survival

To investigate the effects of VCL, L-carnitine, and MIET on SSCs survival, Nanog staining (a specific marker for SSCs) was used. The VCL-sole groups exhibited a significant reduction (P=0.001) in the mean distribution of Nanog+ cells (SSCs) compared to the con-sham group (Fig .6A, B), with this effect developing in a time-dependent manner (P=0.022). L-carnitine and MIET, administered either individually or in combination, significantly increased (P=0.001) the mean number of Nanog⁺ cells per seminiferous tubule compared with the 5-month VCL -only group.

Fig.6.

Alterations in spermatogonial stem cell distribution and self-renewal capacity across experimental groups, as indicated by Nanog expression and repopulation index. A. Nanog immunohistochemistry for spermatogonia stem cells. Decreased Nanog⁺ cells in seminiferous tubules of VCL-sole groups which is increased after L and Exer induction, B. Mean changes in the Nanog⁺ cells per one seminiferous tubule and percentages of tubules with positive repopulation index (RI) in different groups. All data are presented as mean ± SD, and different stars represent significant differences between groups (scale bars: 80 µm). Con-sham; Control-sham, VCL-3; Varicocele-induced after 3 months, VCL-5; Varicocele-induced after 5 months, L; L-carnitine, Exer; Moderate-intensity exercise training, ns; P>0.05, *; P≤0.05, **; P≤0.01, ***; P≤0.001, and ****; P≤0.0001.

The VCL-sole groups (3 and 5 months) also exhibited a significant reduction (P=0.001) in the percentage of tubules with positive RI compared to the con-sham rats. No significant difference (P=0.520) was observed between the 3 and 5-month VCL-sole groups. L-carnitine alone (P=0.001), MIET alone (P=0.009), and their co-administration (P=0.001) significantly increased the percentage of tubules with positive RI compared to the VCL-sole group (5 months).

Discussion

Spermatogenesis is a highly complex biological process regulated by various endocrine factors, growth factors, and cellular mechanisms governing proliferation and differentiation mechanisms, culminating in sperm production within the male gonads. Among the multiple regulatory systems involved in spermatogenesis and spermiogenesis, the self-renewal signaling of SSCs is critical for sperm production (1, 2). This process is partially facilitated by Gdnf-related SSC proliferation through its specific receptors, Gfrα1 and C-ret, forming the Gdnf/Gfrα1/C-ret complex. This complex initiates the expression of Etv5 and Bcl-6b genes, leading to SSC self-renewal (5, 6). Recent animal studies have shown that VCL can disrupt the SSC self-renewal process by interfering with the Gdnf/ Gfrα1/C-ret signaling pathway (16).

L-carnitine, an effective antioxidant, has been demonstrated to protect testicular tissue from oxidative damage (18). Additionally, it has been shown to enhance the effects of exercise and physical activity as well as upregulate the expression of several growth factors (21, 23). MIET, a widely recognized model for improving male reproductive health, has been shown to improve testicular endocrine function, restore antioxidant–oxidant balance, and support SSC self-renewal under stressful conditions (25, 26, 32). Considering these findings, the present study, to the best of our knowledge, is the first to investigate the individual and combined effects of L-carnitine and MIET in a VCL-induced condition. Specifically, this study examines the Gdnf/Gfrα1/C-ret signaling pathway and the expression levels of the self-renewal regulators Etv5 and Bcl-6b. The goal is to determine how these interventions mitigate VCL-induced disruptions in SSC self-renewal, promote overall testicular health, and evaluate the potential synergistic effects of MIET on L-carnitine–mediated protective outcomes.

Gdnf is a glycosylated homodimeric protein that binds to specific Gfrα1 receptors, which are attached to the plasma membrane of SSCs via a glycosyl phosphatidylinositol (GPI) anchor (2). Consequently, Gdnf expressed and secreted by Sertoli cells affects type A-pr and A-S SSCs to initiate self-renewal signaling. In line with previous findings (16), our study demonstrated a significant reduction in Gdnf expression at both mRNA and protein levels under VCL conditions. As previously mentioned, L-carnitine has been shown to upregulate Igf-1, Fgf1, and Ngf expression in various tissues (21-23). Additionally, MIET has been demonstrated to enhance Gdnf expression in Sertoli cells (32). Our findings revealed that both MIET and L-carnitine could upregulate Gdnf expression, with the combined treatment (in the VCL+L+Exer group) showing a more pronounced effect. These preliminary results clearly demonstrate that L-carnitine, at the dose level used in our VCL model, can enhance Gdnf expression in Sertoli cells, similar to its effects on other growth factors such as Igf-1, Fgf1, and Ngf. Furthermore, despite the individual effect of MIET on Gdnf expression, it appears to potentiate the L-carnitine-induced increase in Gdnf expression under experimental VCL conditions.

The Gfrα1 receptor, similar to other members of the Gfrα family (Gfrα2-4), acts as a co-receptor that, upon binding Gdnf, activates the C-ret receptor on the surface of SSCs (2). The binding of the Gdnf/Gfrα1 complex to the extracellular domain of C-ret activates its intracellular tyrosine kinase domain, triggering multiple signaling pathways in SSCs (21). The physiological expression of these receptors in target cells (SSCs and differentiating SSCs/spermatocytes) is essential for maintaining normal spermatogenic function. Disruption of this signaling can adversely affect self-renewal and germ cell differentiation during spermatogenesis (5, 6). Our findings demonstrate that VCL suppresses the expression of Gfrα1 and C-ret in a time-dependent manner at both the mRNA and protein levels. Conversely, L-carnitine treatment, either alone or in combination with MIET, significantly upregulated Gfrα1 and C-ret expression, with the combined treatment producing a more pronounced effect. Although MIET alone showed some improvement, as indicated by IHC cell count analyses, PCR and western blot results revealed that MIET did not significantly enhance Gfrα1 and C-ret expression levels. These findings suggest that while MIET can stimulate Gdnf expression, it is less effective in increasing receptor expression. However, when used concurrently, MIET appears to potentiate the beneficial effects of L-carnitine.

The Bcl-6b and Etv5 genes are downstream transcriptional targets in the Gdnf/Gfrα1/C-ret signaling pathway (21). The expression of these genes, along with others such as ID4 in SSCs, facilitates the self-renewal of type A-S and A-pr SSCs (5, 6). In this study, we assessed the mRNA and protein expression levels of Bcl-6b and Etv5. Our findings revealed that VCL significantly reduced the expression of both genes at both the mRNA and protein levels. However, treatment with L-carnitine and MIET, particularly in combination, significantly upregulated their expression. These findings suggest that the entire Gdnf/Gfrα1/C-ret /Bcl-6b/Etv5 signaling pathway is affected by VCL. While L-carnitine alone can upregulate the expression of all these genes and proteins, MIET primarily enhances Gdnf, Bcl-6b, and Etv5 expression. Additionally, MIET enhances Gfrα1 and C-ret expression when used concurrently with L-carnitine. Importantly, to ensure SSC survival and sufficient SSC presence in the tubules for maintaining spermatogenesis, we utilized Nanog staining (a specific marker for SSCs) and analyzed the RI to assess SSC distribution and regeneration before and after treatment with L-carnitine and MIET in VCL conditions. Our results showed that both L-carnitine and MIET, whether administered alone or together, preserved tubular SSCs and supported their repopulation. Collectively, these findings clearly indicate that L-carnitine and MIET, particularly when co-administered, promote the survival and proliferation of A-s and A-pr SSCs, thereby sustaining spermatogenesis.

The study had several limitations. The reliance on a limited set of markers (Gdnf, Gfrα1, C-ret, Etv5, Bcl-6b) and histological analyses, while informative, may not fully capture all the molecular and cellular changes associated with VCL and the interventions. Additionally, the use of a single dose of L-carnitine and a fixed exercise protocol may not reflect potential variability in outcomes that could arise from different dosages or exercise intensities. Therefore, future studies should investigate the effects of varying L-carnitine doses and diverse exercise regimens to determine optimal therapeutic conditions. At the same time, the strengths of our study are the first combined evaluation of L-carnitine and MIET on the Gdnf/Gfrα1/ C-ret pathway in a VCL model, integration of molecular, histological, and immunohistochemical approaches to validate findings, and demonstration of a synergistic effect of metabolic (L-carnitine) and lifestyle (MIET) interventions. These strengths provide novel insights into potential therapeutic strategies for improving SSC selfrenewal and spermatogenesis under VCL conditions.

Conclusion

This study provides strong evidence that L-carnitine and MIET, both individually and in combination, alleviate VCL-induced disruptions in the self-renewal of SSCs by modulating the Gdnf/Gfrα1/C-ret signaling pathway. The combined administration of L-carnitine and MIET significantly upregulated the expression of key self-renewal regulators, including Gdnf, Bcl-6b, and Etv5, at both the mRNA and protein levels, and enhanced Gfrα1 and C-ret receptor expression more effectively than either treatment alone. Additionally, co-administration of L-carnitine and MIET preserved SSCs within the seminiferous tubules and supported their repopulation, thereby maintaining normal spermatogenesis.

Our findings further confirm that VCL impairs SSC self-renewal by disrupting the Gdnf/Gfrα1/C-ret axis and downregulating critical transcriptional regulators, whereas L-carnitine and MIET can restore this pathway and improve the testicular microenvironment. Importantly, these results underscore the therapeutic potential of combining metabolic support with lifestyle interventions as a promising complementary strategy to mitigate varicocele-associated infertility and preserve long-term male reproductive function.

Conflict of interest

Hossein Nasr-Esfahani and Mazdak Razi are reviewers and editors for this journal. They did not participate in any capacity related to the peer review of this manuscript, nor were they involved in editorial decisions.

Author’s Contributions

Z.I.A.A.-K.; Methodology and Resources. J.T.A.; Conceptualization, Supervision, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Writing-original draft, Writing-review, and Editing. M.H.N.-E.; Conceptualization, Data curation, Investigation, Project administration, Resources, and Visualization. M.R.; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Writing-original draft, Writing-review, and editing. All authors read and approved the final manuscript.