Effects and mechanism of low-intensity pulsed ultrasounds on testicular steroidogenesis in rats

Abstract

Low-intensity pulsed ultrasound (LIPUS) has emerged as a non-invasive therapeutic approach with regenerative potential in various tissues. This study explores its effects on testicular steroidogenesis in male rats. Twenty-four 9-month-old rats were allocated into three groups: control, LIPUS, and LIPUS-M. The LIPUS group underwent pulsed ultrasound therapy targeting the testes twice weekly for 4 weeks, whereas the LIPUS-M group continued with maintenance therapy administered once every 2 weeks for 6 months. At the end of the experiment (16 months), blood and testicular tissues were collected for analysis. Our results showed that LIPUS therapy significantly elevated serum testosterone level and increased the number of Leydig cells (LCs), as indicated by steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) immunofluorescence. Collagen IV staining showed increased interstitial vascularization, and senescence-associated β-galactosidase (SA-β-Gal) staining suggested reduced cellular senescence. Although the LIPUS-M group also showed increased LC counts and angiogenesis, serum testosterone levels were not elevated. These findings demonstrate that LIPUS can activate LCs and enhance endogenous testosterone production, supporting its potential as a noninvasive regenerative approach for hypogonadism.

INTRODUCTION

Hypogonadism, characterized by low serum testosterone levels and associated symptoms, affects a significant proportion of the male population. Its prevalence ranges from 16% to as high as 62%, depending on the population studied. It is most common among aging men with comorbidities such as obesity, metabolic syndrome, and chronic illness.1,2,3,4 Testosterone plays a critical role in multiple physiological processes, including the maintenance of muscle mass, energy metabolism, and physical function.5,6,7 It stimulates protein synthesis in muscle tissues, enhances muscle contractility, and supports overall vitality and motivation. Declining testosterone levels are associated with reduced muscle mass and strength, increased fat accumulation, and decreased physical activity and can lead to sarcopenia and frailty in males.

Testosterone replacement therapy (TRT) has long been the cornerstone of clinical management.8,9,10 TRT can alleviate many of the symptoms associated with hypogonadism, but its long-term use is not without drawbacks. Exogenous testosterone suppresses the hypothalamic–pituitary–gonadal axis and impairs the function of testicular Leydig cells (LCs), rendering the individual dependent on ongoing therapy. Moreover, concerns about cardiovascular risks have historically limited the broad application of TRT. Although a recent meta-analysis involving over 138 000 men reported that TRT may reduce all-cause mortality compared to no treatment, its requirement for lifelong administration and routine monitoring remains a clinical concern.11 Nevertheless, its requirement for lifelong administration, routine monitoring, and potential adverse effects continue to prompt the search for alternative strategies. An ideal approach would involve stimulating the endogenous testosterone production machinery, particularly LCs, rather than replacing testosterone exogenously.

Recent studies have revealed that adult testes retain a population of stem Leydig cells (SLCs) that possess the potential to proliferate and differentiate into mature LCs under appropriate stimulation.12,13,14 These endogenous progenitor cells are thought to play an important role in maintaining or restoring testosterone production following injury or aging. Therefore, activating such intrinsic regenerative mechanisms has emerged as a promising strategy for restoring physiological androgen levels.

Low-intensity pulsed ultrasound (LIPUS) is a non-invasive biophysical therapy that has been widely applied in regenerative medicine.15 Previous studies have shown that LIPUS enhances tissue regeneration not only in musculoskeletal and neural tissues15 but also in hormonal organs such as the testes and thyroid.16,17 Notably, in vitro experiments have demonstrated that LIPUS can stimulate Leydig cell proliferation and upregulate steroidogenic enzymes, suggesting that it may enhance testicular steroidogenic function through activation of local progenitor or supporting cell populations.17

Based on this, we hypothesized that LIPUS could serve as a novel, non-pharmacological method to restore endogenous testosterone production by targeting testicular stem cells. In this study, we evaluated the effects and mechanism of LIPUS on testicular steroidogenesis in rats, focusing on testosterone synthesis, LC proliferation, vascularization, and cellular senescence. The experimental workflow was designed to assess both short-term and long-term effects of LIPUS, offering insight into its potential as a sustainable and physiological alternative to TRT.

MATERIALS AND METHODS

Animals and experimental design

Adult male Sprague Dawley rats (9 months old) were used in this study. All animals were housed under standard laboratory conditions (12-h light/dark cycle, 22°C–24°C, and 50%–60% humidity) with free access to food and water. A total of 24 animals were randomly assigned into three groups (n = 8 per group). The control (Ctrl) group received no therapy. The LIPUS group underwent LIPUS therapy twice a week for 4 weeks. The LIPUS-M group underwent the same initial LIPUS regimen as the LIPUS group, followed by a maintenance therapy once every 2 weeks for an additional 6 months (Figure 1a). All rats were anesthetized with inhaled isoflurane throughout the LIPUS procedure to minimize stress. Identical anesthesia and handling protocols were applied to all three groups to exclude potential confounding effects of restraint or anesthesia on hormonal measurements. Serum testosterone levels were evaluated at baseline (before treatment), T1 (after 4 weeks of LIPUS treatment), and T2 (after the 6-month maintenance phase). Serum luteinizing hormone (LH) levels were evaluated at baseline and T2. All rats were sacrificed at 16 months of age for endpoint evaluation (end of experiment [EOE]). All experiments were approved by the Institutional Animal Care and Use Committee at the University of California (San Francisco, CA, USA; Approval No. AN208836-00).

Figure 1.

Experimental design and effects of LIPUS on serum testosterone and LH levels in rats. (a) Schematic illustration of the experimental workflow. Nine-month-old male rats were randomly assigned to three groups: Ctrl, LIPUS, and LIPUS-M. The Ctrl group received sham treatment. The LIPUS group received low-intensity pulsed ultrasound (67 mW cm⁻², 1.7 MHz, 3 min) twice per week for 4 weeks. The LIPUS-M group received the same initial treatment, followed by maintenance stimulation once every 2 weeks for 6 months. Serum testosterone levels were evaluated at baseline, T1, and T2. Serum LH levels were evaluated at baseline and T2. All rats were sacrificed at 16 months of age for endpoint evaluation (EOE). (b) Significant increases of serum testosterone levels were observed in the LIPUS group at T1 and T2 compared to the baseline (^*P < 0.05, ^**P < 0.01). Meanwhile, the LIPUS group showed a significant increase compared to the Ctrl group at T2 (^#P < 0.05). (c) LH levels were significantly reduced in the LIPUS-M group compared to the Ctrl group at T2 (^*P < 0.05). Data are presented as box-and-whisker plots. Ctrl: control; LIPUS: low-intensity pulsed ultrasound; LH: luteinizing hormone; EOE: end of experiment; T1: after 4 weeks of LIPUS treatment; T2: after the 6-month maintenance phase.

LIPUS treatment protocol

LIPUS (Beijing Wanboli Medical Device, Inc., Beijing, China) was applied using a therapeutic ultrasound device operating at a frequency of 1.7 MHz and a spatial average intensity of 67 mW cm⁻². The probe was gently placed in contact with the testicular region using ultrasound coupling gel to ensure consistent energy transmission. Each session lasted for 3 min. Animals in the LIPUS and LIPUS-M groups received identical treatment parameters for the first 4 weeks (twice per week), while the LIPUS-M group additionally received maintenance stimulation once every 2 weeks for 6 months. Control animals underwent identical handling and anesthesia without ultrasound activation (sham treatment).

Blood sampling and experimental timing

Blood samples were collected from the tail vein at three time points: baseline, T1, and T2. To minimize stress, all sampling was performed under inhaled isoflurane anesthesia, and the same procedures were applied to all groups, including the Ctrl group. To control for circadian variation in gonadotropin and testosterone secretion, sampling was consistently performed in the morning (8:00 a.m.–10:00 a.m.). Blood was collected 24 h after the final LIPUS session at each time point to avoid acute effects of ultrasound exposure. All rats were 9 months old at the beginning of the experiment, representing sexually mature adults with stable endocrine profiles.

At the end of the experimental period (16 months of age), all animals were euthanized under anesthesia. Blood was collected for final hormone analysis, and both testes were harvested and fixed for histological and immunofluorescence evaluation. The experimental endpoint was defined as the EOE.

Hormone measurement

Serum testosterone (R&D Systems, Inc., Minneapolis, MN, USA) and LH (Enzo Life Sciences, Farmingdale, NY, USA) levels were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. All samples were run in duplicate.

Immunofluorescence staining and cell quantification

Paraffin-embedded testicular sections were used for immunofluorescence staining. Primary antibodies against steroidogenic acute regulatory protein (STAR; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD; Santa Cruz Biotechnology Inc.), DEAD-box helicase 4 (DDX4; Santa Cruz Biotechnology Inc.), and collagen IV (Abcam Inc., Waltham, MA, USA) were used to evaluate LCs, germ cells, and vascular structures. 4’,6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Fluorescence intensity of STAR (per high-power field [HPF]) and LC counts (per low-power field [LPF]) were quantified using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA). The numbers of blood vessels and senescent cells (assessed via collagen IV and senescence-associated β-galactosidase [SA-β-Gal] staining) per HPF were also quantified.

Statistical analyses

Data were statistically analyzed using SPSS 20.0 (IBM Corp., Armonk, NY, USA). Normally distributed continuous variables were expressed as the mean ± standard deviation (s.d). Statistical comparisons among groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or nonparametric tests followed by Mann–Whitney U tests based on whether the data meet the assumption of homogeneity of variance. P < 0.05 was considered statistically significant.

RESULTS

LIPUS increases serum testosterone and suppresses LH levels

To evaluate the endocrine effects of LIPUS on systemic testosterone production, serum hormone levels were measured in each group. As shown in Figure 1b, serum testosterone levels were significantly elevated in the LIPUS group compared to the baseline at T1 and T2 (P < 0.05 and P < 0.01, respectively). The LIPUS-M group demonstrated a moderate increase that did not reach statistical significance at T1. Meanwhile, the LIPUS group showed a significant increase compared to the Ctrl group at T2 (P < 0.05). In contrast, serum LH levels were significantly reduced in LIPUS-M-treated groups compared to the Ctrl group at T2 (P < 0.05; Figure 1c). This inverse relationship suggests that LIPUS-M group-mediated testosterone elevation exerts a negative feedback effect on pituitary LH secretion.

LIPUS enhances STAR expression and increases LC number in testicular tissue

To further investigate the effects of LIPUS on steroidogenic activity in the testis, we performed immunofluorescence staining for STAR, the key regulator of testosterone synthesis, in testicular sections from three groups: Ctrl, LIPUS, and LIPUS-M. As shown in Figure 2a, STAR expression was markedly increased in the interstitial regions of the LIPUS group compared to the Ctrl group. Quantitative analysis confirmed a significant increase in STAR fluorescence intensity in the LIPUS group compared to the Ctrl group (P < 0.05), while the LIPUS-M group showed a mild but non-significant increase (Figure 2b). Furthermore, the absolute number of LCs per LPF was significantly elevated in both the LIPUS and LIPUS-M groups relative to the Ctrl group (both P < 0.01; Figure 2c), suggesting that LIPUS stimulation promotes LC proliferation or survival.

Figure 2.

LIPUS enhances STAR expression and increases LC number in testicular tissue. (a) Representative immunofluorescence images of STAR (LC marker, red), DDX4 (green), and DAPI nuclear staining (nuclei, blue) in testis sections from the Ctrl, LIPUS, and LIPUS-M groups. The differences of treatments among the groups are shown in the legend part of Figure 1. (b) Quantification of STAR fluorescence intensity in testicular sections, showing a significant increase in the LIPUS group compared to the Ctrl group (^*P < 0.05), while no significant difference exists between LIPUS and LIPUS-M groups. (c) Absolute number of Leydig cells per low-power field (LPF), indicating significantly more LCs in both LIPUS and LIPUS-M groups compared to the Ctrl group (^**P < 0.01). Ctrl: control; LIPUS: low-intensity pulsed ultrasound; STAR: steroidogenic acute regulatory protein; DDX4: DEAD-box helicase 4; DAPI: 4´-6-diamidino-2-phenylindole; LCs: Leydig cells; LPF: low-power field.

LIPUS promotes angiogenesis in testicular tissue

In parallel, staining for collagen IV demonstrated enhanced vascular structures in the interstitial space, particularly in the LIPUS group. Statistical analysis showed a significant increase in blood vessel number per HPF in both LIPUS-treated groups compared to the Ctrl group, with the highest vascular density observed in the LIPUS group (P < 0.01, LIPUS vs Ctrl; P < 0.01, LIPUS vs LIPUS-M; Figure 3). These results indicate that LIPUS promotes angiogenesis, which may contribute to the improved LC function and testosterone synthesis.

Figure 3.

LIPUS promotes angiogenesis in testicular interstitial tissue. (a) Immunofluorescence staining for 3β-HSD (red) and Col-IV (basement membrane/blood vessel marker, green) to assess vasculature. (b) Quantification of blood vessel number per HPF, indicating that LIPUS significantly enhances angiogenesis in testicular tissue compared to the Ctrl and LIPUS-M groups. ^**P < 0.01. The differences of treatments among the groups are shown in the legend part of Figure 1. Col-IV: collagen IV; Ctrl: control; LIPUS: low-intensity pulsed ultrasound; 3β-HSD: 3β-hydroxysteroid dehydrogenase; HPF: high-power field.

LIPUS attenuates LC senescence in testicular tissue

To evaluate the effect of LIPUS on LC senescence, SA-β-Gal staining was performed on testicular sections from Ctrl, LIPUS, and LIPUS-M groups. As shown in Figure 4a–4c, a substantial number of senescent cells were observed in the interstitial compartment of the testes in the Ctrl group. In contrast, a reduced number of SA-β-Gal-positive cells were found in the LIPUS-M group, while the LIPUS group exhibited the fewest senescent cells.

Figure 4.

LIPUS reduces senescent cell accumulation in testicular tissue. Representative images of SA-β-Gal staining in testis sections from the (a) Ctrl, (b) LIPUS, and (c) LIPUS-M groups. The differences of treatments among the groups are shown in the legend part of Figure 1. Senescent cells are indicated by blue staining (arrows). Images were captured at 200× magnification. (d) Quantification of senescent cell absolute number per HPF. Both LIPUS-M and LIPUS groups showed significantly reduced numbers of senescent cells compared to the Ctrl group, with the lowest levels observed in the LIPUS group (LIPUS vs LIPUS-M). Ctrl: control; LIPUS: low-intensity pulsed ultrasound; SA-β-Gal: senescence-associated β-galactosidase; HPF: high-power field.

Quantification of senescent cells per HPF revealed a significant reduction in the number of SA-β-Gal-positive cells in the LIPUS group compared to both the Ctrl and LIPUS-M groups (both P < 0.01), as depicted in Figure 4d. These results indicate that LIPUS treatment effectively delays LC senescence, with LIPUS exhibiting superior anti-senescent effects compared to extended maintenance exposure.

DISCUSSION

In this study, we demonstrated that LIPUS significantly enhances endogenous testosterone production in rats through multiple mechanisms, including activation of LCs, reduction of testicular senescence, and improved testicular vascularization. These findings support the hypothesis that LIPUS is a promising, non-invasive therapeutic modality for restoring physiological testosterone synthesis in males, potentially offering a safer and more sustainable alternative to conventional TRT.

Interestingly, while the LIPUS-M group also exhibited increased LC numbers and vascular density, serum testosterone levels were not elevated. This discrepancy may be attributed to an endocrine feedback mechanism. The prolonged stimulation induced by LIPUS-M likely triggers continuous testosterone high output, which in turn suppresses LH secretion via the hypothalamic–pituitary–gonadal axis.18 The resulting decline in LH impairs endogenous testosterone synthesis, counteracting the otherwise beneficial cellular and vascular effects of LIPUS-M (Figure 5).

Figure 5.

Proposed mechanism of LIPUS and LIPUS-M effects on testicular steroidogenesis. LIPUS treatment promotes LC proliferation, enhances interstitial angiogenesis, and delays LC senescence, collectively contributing to increased testosterone synthesis. In contrast, long-term maintenance LIPUS treatment (LIPUS-M), while similarly enhancing LC number and vascular density, results in a negative feedback loop via the hypothalamic–pituitary–gonadal axis. The sustained elevation of T levels leads to suppression of LH secretion from the hypophysis, ultimately reducing endogenous testosterone production despite local testicular changes. LIPUS: low-intensity pulsed ultrasound; LCs: Leydig cells; LH: luteinizing hormone; T: testosterone.

LIPUS is an emerging non-invasive therapeutic modality that has shown efficacy in the treatment of a wide range of diseases.15,16,17 Clinical studies have demonstrated that LIPUS can accelerate bone healing, enhance angiogenesis, and stimulate osteogenic and chondrogenic differentiation, particularly in delayed union or nonunion fractures and soft-tissue injuries.19,20 In addition to promoting bone regeneration, LIPUS has shown beneficial effects on cartilage repair, tendon and ligament healing, and postoperative recovery by modulating inflammation and mechanotransductive signaling pathways.21,22 The overall evidence confirms that LIPUS is a safe, noninvasive modality capable of activating tissue-resident progenitor cells and improving microvascular remodeling. These findings provide strong mechanobiological and translational rationale for extending LIPUS applications beyond orthopedics to endocrine and reproductive organs, such as the testis, for restoring local regenerative and steroidogenic functions.

Our immunofluorescence analysis revealed an upregulation of STAR expression, key rate-limiting factor in steroidogenesis, in testicular tissue of LIPUS-treated rats. These findings suggest that LIPUS not only enhances LC steroidogenic activity but also promotes the proliferation and/or differentiation of LCs, possibly through activation of SLCs.12,13,14 This highlights the potential of LIPUS to engage intrinsic regenerative mechanisms within the testis.

Testicular dysfunction is often compounded by cellular senescence and impaired microcirculation.16,23 Our results showed a clear reduction in senescent cell burden, as indicated by decreased SA-β-Gal-positive cells in the LIPUS group, suggesting that LIPUS may exert anti-senescent effects in the testicular microenvironment. Furthermore, the significant increase in blood vessel density observed in LIPUS-treated animals indicates improved vascular supply, which is essential for nutrient delivery, hormone transport, and the maintenance of a healthy testicular tissue.

Notably, the LIPUS-M group did not achieve the desired results. Although the LIPUS-M regimen involved a longer maintenance treatment than the LIPUS group and also promoted the increase in the number of LCs and interstitial blood vessels, it failed to elevate testosterone levels. According to our study, this may be related to a reduction in LH levels caused by prolonged LIPUS stimulation.18,24,25 Therefore, a 6-month maintenance course of LIPUS treatment does not appear to yield better outcomes.

The absence of further testosterone elevation in the LIPUS-M group, initially attributed to hypothalamic–pituitary feedback suppression of LH, remains a plausible yet unproven mechanism. Beyond this systemic endocrine regulation, prolonged or repetitive ultrasonic exposure may induce local tissue adaptation or desensitization, thereby attenuating the mechanotransductive and steroidogenic responsiveness of LCs. Although direct evidence in testicular tissue is lacking, studies in other regenerative contexts have shown that the efficacy of LIPUS may plateau or decline when stimulation extends beyond the optimal duration.21,22,26 These studies emphasize that the biological effects of LIPUS are highly dependent on treatment parameters – particularly frequency, intensity, and duration – and that excessive or prolonged exposure may reduce cellular responsiveness through adaptive mechanisms. A study examining LIPUS effects on bone integration of dental implants reported that the optimal window for new bone formation occurs during the early healing phase (approximately 4 weeks), after which further stimulation confers minimal additional benefit.27

To date, few studies have specifically examined “maintenance-phase” LIPUS therapy, making our investigation an exploratory attempt to address this gap. Our findings suggest that LIPUS efficacy may follow a time-dependent saturation pattern rather than a simple linear increase with longer treatment duration.

Taken together, our findings indicate that short-term LIPUS can effectively enhance LC activity and vascular remodeling, whereas prolonged maintenance exposure offers no additional endocrine advantage. This highlights that LIPUS therapy, when translated to clinical use, should prioritize the optimization of treatment duration and frequency rather than indefinite or repeated maintenance regimens. By defining an appropriate, time-limited course, LIPUS may achieve maximal endocrine benefit without unnecessary overtreatment, thereby providing a rational foundation for future clinical trial design and individualized therapeutic strategies in male reproductive endocrinology.

This study has several limitations. First, although our findings suggest activation of SLCs, the precise cellular origin of the observed regenerative effects remains unclear, as definitive lineage-tracing experiments were not performed. Second, the safety profile and optimal treatment parameters of LIPUS in humans have yet to be determined. Third, caution is required when extrapolating rodent data to human physiology, underscoring the need for future translational and clinical investigations. Fourth, the relatively small sample size (n = 8 per group) and the absence of a priori power calculation may limit the statistical robustness and generalizability of our results. Larger studies with formal power estimation are therefore warranted to validate these preliminary observations. In addition, at the T1 time point, both the LIPUS and LIPUS-M groups showed an overall increase in serum testosterone; however, statistical significance was achieved only in the LIPUS group. This discrepancy is most likely due to interindividual variability rather than true biological divergence, as both groups received identical ultrasound exposure during this phase and no significant differences were detected between them. Finally, LH concentrations were measured only at baseline and at T2, not at T1. Therefore, the proposed feedback interaction between LH and testosterone remains speculative and requires more detailed temporal profiling in future studies.

In conclusion, our study provides compelling evidence that LIPUS stimulates testosterone synthesis through activation of LCs, suppression of testicular senescence, and promotion of interstitial angiogenesis. These findings open new avenues for the non-invasive treatment of testosterone deficiency and highlight the potential of LIPUS as a regenerative strategy for male reproductive aging.

AUTHOR CONTRIBUTIONS

TFL and GL designed and supervised the research. XM, FZ, and CQ analyzed the data and wrote the manuscript. XM, FZ, MHW, GW, TFL, and GL collected the data and revised the manuscript. FZ, WW, GW, LB, and GL interpreted the data. XM, FZ, MHW, CQ, GW, LB, TFL, and GL conducted the LIPUS treatment. All authors read and approved the final manuscript.

COMPETING INTERESTS

Guiting Lin is the editorial board member of the journal and was not involved in the peer review or the decision making of the article. Other authors declare no competing interests.