Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway.

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INTRODUCTION

Leydig cells (LCs) are the primary site for the synthesis and secretion of testosterone. 1 , 2 Testosterone promotes the development of male secondary sexual characteristics, maintains normal libido, and also promotes the growth of sperm and male reproductive organs. 3 Currently, cyclophosphamide (CP) is a widely used anti-tumor drug and immunosuppressant that is often combined with other chemotherapy drugs to treat malignant tumors, such as leukemia, lymphoma, and multiple myeloma. 4 , 5 It is also used to prevent graft-versus-host disease and treat autoimmune disorders such as systemic lupus erythematosus, childhood nephrotic syndrome, and idiopathic thrombocytopenic purpura. 6 , 7 However, CP also causes toxic effects on LCs, resulting in the diminished ability of LCs to synthesize testosterone and ultimately causing testosterone deficiency (TD). 8 , 9 The St. Jude Lifetime Cohort Study revealed that over 50% of children treated with CP develop oligozoospermia or azoospermia in adulthood, and approximately 21.6% of the patients develop TD. 10 , 11 For patients with TD, testosterone replacement therapy (TRT) is the preferred treatment method recommended by the American Urological Association (AUA) and European Association of Urology (EAU) guidelines. 12 Although TRT can effectively improve the symptoms caused by TD, it also causes various adverse reactions. 13 Therefore, further research is required to develop a more effective therapeutic approach against cyclophosphamide-induced testosterone deficiency (CPTD).

In the 21 st century, stem cell therapy has emerged as a research hotspot for treating diseases of the male reproductive system. Bone marrow mesenchymal stem cells (BMSCs) transplantation for various disease therapies depends on their differentiation ability and paracrine function. 14 Nevertheless, after transplantation of BMSCs into recipients, the stem cells are less likely to differentiate directly into tissues owing to the influence of the transplanted microenvironment, but mainly play reparative and therapeutic roles through paracrine function. 15 Exosomes (exos) are considered to be the potent secretory components of BMSCs, which play a vital role in intercellular communication, such as transferring functional proteins, metabolites, and nucleic acids to recipient cells. 16 , 17 Further, they involve in numerous cellular physiological processes, including cell signal transduction, cell metabolism, cell autophagy, and immunity regulation. 18 , 19 We previously reported that BMSCs-exos could ameliorate CP-induced spermatogenesis dysfunction via inhibiting the p38 mitogen-activated protein kinase (p38-MAPK) signaling pathway and testicular ischemia–reperfusion. 20 , 21 These studies suggested that BMSCs-exos play an indispensable role in treating male reproductive disorders. However, their role and specific mechanism in CPTD remains unexplored.

Autophagy is the central intracellular degradation mechanism dependent on the lysosomal pathway, and can degrade or reuse proteins and damaged organelles that accumulate in the cytoplasm. 22 , 23 In terms of the protection of cell homeostasis, there is crosstalk between exosomes and autophagy. 19 Several recent studies have shown that mesenchymal stem cell (MSC)-derived exos regulate cell autophagy and have therapeutic potential for diseases such as type 2 diabetes, diabetic nephropathy, stroke, liver fibrosis, and osteoarthritis. 24 – 28 Autophagy is highly active in the testicular interstitium and is closely associated with testosterone synthesis in LCs. 29 – 32 Gao et al . 33 observed that autophagy promotes cholesterol uptake to regulate testosterone synthesis in LCs. However, whether BMSCs-exos could ameliorate CPTD by regulating autophagy in LCs remains largely unknown.

In the present study, we aimed to establish CPTD models in vivo and in vitro . We also assessed the role of CP in regulating autophagy in LCs. Finally, we investigated whether BMSCs-exos could enhance autophagy and facilitate testosterone synthesis in LCs, thereby ameliorating CPTD. Overall, the findings of our study might provide a novel therapeutic approach for alleviating CPTD.

MATERIALS AND METHODS

Animal and animal ethics

C57BL/6 male mice of 8 weeks (Ruige Biotechnology Co., Ltd., Guangzhou, China) were randomly separated into four groups (6 mice per group): control (normal saline, intraperitoneal injection), CP (50 mg kg −1 per day, 7 days, intraperitoneal injection; GLPBIO, Montclair, CA, USA), CP (combination) + BMSC-exo (100 μg ml −1 , 0.1 ml per day, 7 days, intravenous injection), and CP (combination) + BMSC-exo (combination) + 3-MA (15 mg kg −1 per day, 7 days, intraperitoneal injection). After the drug combinations were administered, the mice were housed normally for 2 weeks. Later, all mice were weighed and sacrificed. The bilateral testes, as well as serum, were collected and stored for subsequent experiments. Before conducting all experimental animal protocols in this study, we obtained approval from the Laboratory Animal Ethics Committee of Southern Medical University (Guangzhou, China; Approval No. SMUL2022082).

Isolation, culture, and characterization of BMSCs

After the mice were sacrificed, the femurs and tibias were aseptically harvested for BMSCs isolation. The bone marrow cavity was flushed with mouse BMSC complete medium (Procell, Wuhan, China). The obtained bone marrow washing fluid was centrifuged (L550, Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China) at 500 g for 5 min, resuspended in mouse bone marrow mesenchymal stem cell complete medium, and the cells were inoculated into a Petri dish and cultured at 37°C in a humidified incubator containing 5% CO 2 . After 72 h of culture, nonadherent cells were removed by refreshing the cell culture medium, and the remaining adherent cells were cultured until they reached 80% confluence. Following the digestion of adherent cells with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco, Grand Island, NY, USA), the cells were passaged. BMSCs at passages 3–5 (P3–5) were used in subsequent experiments.

A 100-mesh filter was used to filter the cell suspension obtained from digestion with 0.25% trypsin-EDTA of BMSCs at P3. A flow cytometry analysis was also performed to detect the surface marker proteins of BMSCs following the mesenchymal stem cell surface marker detection kit (OriCell, Guangzhou, China) manufacturer’s instructions.

BMSCs at P3 were cultured with osteoblast- and adipocyte-inducing medium. After 14–21 days, the differentiated osteoblasts and adipocytes were stained with Alizarin Red (OriCell) and Oil Red O (OriCell), respectively, and further observation was performed under a microscope (Olympus, Tokyo, Japan).

Isolation and identification of BMSCs-exos

After BMSCs density reached approximately 80%, BMSC complete medium was replaced with a serum-free BMSC medium, and the cells were cultured for 48 h. The specific extraction and preservation methods of BMSCs-exos are based on our previous research. 20 The transmission electron microscopy (TEM; Philips-Tecnal, Amsterdam, The Netherlands) was used to observe the morphology of BMSCs-exos. The ZetaView system (Particle Metrix, Munich, Germany) measured the BMSCs-exos concentrations and sizes. Western blot was used to identify the BMSCs-exos surface protein markers.

TM3 mouse Leydig cells

The cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were incubated in TM3 mouse Leydig cell complete medium (Procell), and cultured at 37°C in a humidified incubator containing 5% CO 2 .

BMSCs-exos uptake assay

The cytoskeleton and nucleus of TM3 mouse Leydig cells were stained with phalloidin (Sigma, St. Louis, MO, USA) and 4’,6-diamidino-2-phenylindole (DAPI; Sigma), respectively. PKH-67 (Sigma)-labeled exosomes were co-cultured with TM3 mouse Leydig cell culture medium for 12 h. Subsequently, the cellular uptake of BMSCs-exos was observed with an inverted microscope (Olympus).

Cell Counting Kit-8 (CCK-8) assay

A CCK-8 kit (Fude Biotechnology Co., Ltd., Hangzhou, China) was used to determine TM3 mouse Leydig cell viability following the instructions provided by the manufacturer. A microplate reader (BioTek, Winooski, VT, USA) was used to measure absorbance at 450 nm.

Testosterone measurement

Serum testosterone and cell supernatant concentrations were determined by enzyme-linked immunosorbent assay (ELISA; Mlbio, Shanghai, China) in accordance with the manufacturer’s instructions. An ELISA reader (BioTek) was used to measure optical density (OD) at 450 nm in each well.

Quantitative real-time PCR (qPCR)

Total RNA from the TM3 mouse Leydig cell extract was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The optical density (OD) ratio of absorbance at 260 nm and 280 nm (A260/A280) was used to determine RNA purity; RNA with a purity between 1.8 and 2.0 was used in this study. A reverse transcription system kit (Vazyme, Nanjing, China) was used to synthesize complementary DNA (cDNA). We performed qPCR using a StepOnePlus (ABI, Carlsbad, CA, USA). The ChamQ Universal SYBR qPCR Master Mix kit (Vazyme) was used to perform qPCR under the established thermal cycling conditions (predenaturation: 95°C for 30 s, cycle 1; cyclic reaction: 95°C for 10 s, 60°C for 30 s, cycle 40; melting curve: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, cycle 1). The primer sequences for the targeted genes used in this study are shown in Supplementary Table 1 .

Immunofluorescence staining

The collected mouse testes were fixed with the animal testis tissue fixative (Servicebio, Wuhan, China), dehydrated, embedded in paraffin, and cut into 4-μm sections. When TM3 mouse Leydig cells reached confluence, they were exposed to 4% paraformaldehyde (Beyotime, Shanghai, China) for fixing at room temperature (20°C–25°C) for 20 min and exposed to 0.25% Triton X-100 in phosphate-buffered saline (PBS; Gibco) for permeabilizing at room temperature for 15 min. The sections and cells were then incubated with diluted antibodies against microtubule-associated protein 1A/1B-light chain 3 (LC3; Abcam, Cambridge, UK) and 3β-hydroxysteroid dehydrogenase (3β-HSD; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Subsequently, the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Bioss, Beijing, China) were added and incubated at room temperature for 90 min. Nuclear counterstaining was carried out by staining the sections and cells with DAPI after washing them with PBS. Images were captured at ×200 using an inverted microscope (Olympus). The mean fluorescence intensities of LC3 and 3β-HSD were determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Western blot

Western blot was performed based on a previously described protocol. 20 Proteins were extracted from cells using a radioimmunoprecipitation assay lysis buffer (RIPA; Biosharp, Hefei, China) that contained protease and phosphatase inhibitors (APExBIO, Houston, TX, USA). Bicinchoninic acid assay (BCA) protein quantification kit (Meilunbio, Dalian, China) was used to quantify the extracted cellular protein. An electrophoresis gel (Biosharp) containing sodium dodecyl sulfate and polyacrylamide was used for electrophoresis. The total protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Biosharp) and blocked with 5% bovine serum albumin (BSA; Elabscience, Wuhan, China) at room temperature for 1 h. Subsequently, the membrane was incubated with diluted antibodies against LC3 (Proteintech, Wuhan, China), Beclin-1 (Proteintech), P62 (Proteintech), steroidogenic acute regulatory protein (StAR; Proteintech), recombinant cytochrome P450 11A1 (CYP11A1; Proteintech), adenosine monophosphate-activated protein kinase (AMPK; CST, Boston, MA, USA), photophosphorylation-AMPK (p-AMPK; CST), mTOR (CST), photophosphorylation-mTOR (p-mTOR; CST), and β-actin (Proteintech) at 4°C overnight. On the 2 nd day, a 1-h incubation with the corresponding secondary antibodies was carried out at room temperature on PVDF membranes. The protein bands were visualized with electrochemiluminescence (ECL) detection reagent (NCM Biotech, Suzhou, China). The density of the protein bands was measured with ImageJ software.

Statistical analyses

In all experiments, the data were collected independently three times, and the results were presented as mean ± standard deviation (s.d.). Statistical analyses were conducted with SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) with one-way analysis of variance (ANOVA). Histograms were constructed using GraphPad Prism software version 8 (GraphPad Software Inc., San Diego, CA, USA). P < 0.05 designates a statistically significant difference.

RESULTS

Identification of BMSCs and BMSCs-exos

As shown in Figure 1a , the morphology of BMSCs was diverse, with clear boundaries and good refraction. BMSCs were identified by their differentiation into osteoblasts and adipocytes ( Figure 1b ). Flow cytometry analysis demonstrated that the surface markers CD29, CD44, CD34, and Ly-6AE of BMSCs were all positive, with a positivity rate of more than 90.0%; however, the cells were negative for CD31 and CD117 expression ( Figure 1c ). BMSCs-exos appeared as circular vesicles with bilayer membranes ( Figure 1d ). Nanoparticle tracking analysis (NTA) revealed that the diameter of these vesicles was in the range of 30–150 nm ( Figure 1e ). Compared to the expression in BMSCs, surface markers CD63 and TSG101 were also highly expressed in BMSCs-exos, but that of calnexin was negligible ( Figure 1f ).

BMSCs-exos ameliorate CP-induced TD via enhancing LCs autophagy in vivo

LCs play a vital role in promoting spermatogenesis and male reproductive organ development, as well as in maintaining male secondary sexual characteristics and sexual function by regulating testosterone synthesis and secretion. Autophagy is involved in regulating testosterone biosynthesis in LCs. We established a CP-induced TD mouse model and explored whether BMSCs-exos could improve CP-induced low testosterone levels by enhancing autophagy in LCs. The results imply that CP significantly suppressed antigen expression of LC3 and 3β-HSD in the testis interstitium when compared with the control group ( P < 0.001). In contrast, BMSC-exo supplementation reversed the expression of LC3 and 3β-HSD, which was counteracted by 3-MA supplementation ( Figure 2a – 2d ). We also observed that CP caused a significant decrease in serum testosterone levels, whereas supplementation with BMSCs-exos improved these levels. However, the improved testosterone levels were counteracted by the 3-MA treatment ( Figure 2e ). These results collectively suggest that BMSCs-exos may promote cell autophagy in CP-exposed LCs and further ameliorate their ability to synthesize testosterone.

BMSCs-exos inhibit cell death in CP-exposed TM3 mouse LCs

We previously reported that BMSCs-exos could penetrate the blood–testis barrier and be taken up by germ cell 1 spermatogonial (GC1-spg) cells. 20 In this study, we also observed that TM3 mouse LCs could uptake BMSCs-exos efficiently ( Figure 3a ). To establish an optimal injury model, TM3 mouse LCs were exposed to different concentrations of CP (0 μg ml −1 , 5.0 μg ml −1 , 10.0 μg ml −1 , 20.0 μg ml −1 , 30.0 μg ml −1 , and 40.0 μg ml −1 ) for 24 h. As measured by the CCK-8 assay, there was a dose-dependent reduction in cell viability, beginning with a dosage of 10.0 μg ml −1 . However, this attenuation was the most apparent at 20.0 μg ml −1 ( Figure 3b ). These results collectively indicated that CP at a dose of 20.0 μg ml −1 was the optimal concentration to establish the injury model. Subsequently, we used different concentrations of BMSCs-exos (0 μg ml −1 , 50 μg ml −1 , 100 μg ml −1 , and 200 μg ml −1 ) to treat LCs for 24 h. The results revealed that cell viability was evidently increased at a dosage of 100 μg ml −1 compared to that at other concentrations ( Figure 3c ). Based on these data, we used this concentration of BMSCs-exos to treat LCs in the subsequent experiments. BMSCs-exos could effectively alleviate cell apoptosis in CP-exposed TM3 mouse LCs, as observed by flow cytometry analysis. 34 Western blot analysis revealed that BMSCs-exos significantly elevated the anti-apoptotic Bcl-2 protein expression and reduced the pro-apoptotic Bax protein expression ( P < 0.01; Figure 3d and 3e ).

BMSCs-exos promote autophagy in CP-exposed TM3 mouse LCs

After the CP-induced TD model was established in vitro , BMSCs-exos were added for co-culture for 24 h. We further examined the level of autophagy in TM3 mouse LCs. The qPCR and western blot analyses demonstrated that CP significantly decreased the expression of LC3 and Beclin-1 and also elevated the expression of p62 in TM3 mouse LCs compared to that in the control group ( P < 0.01). After the BMSC-exo supplement, LC3 and Beclin-1 were upregulated, while P62 was downregulated ( Figure 4a – 4c ). Immunofluorescence staining further confirmed that CP decreased LC3 protein expression in TM3 mouse LCs, and BMSCs-exos increased the expression of LC3 in these cells ( P < 0.01; Figure 4d and 4e ). However, the ability of BMSCs-exos to regulate autophagy was reversed by 3-MA treatment.

BMSCs-exos improve the testosterone synthesis in CP-exposed TM3 mouse LCs

Next, we examined the ability of TM3 mouse LCs to synthesize testosterone. The qPCR and western blot analyses indicated that CP significantly decreased StAR and CYP11A1 expression in TM3 mouse LCs compared with that in the control group ( P < 0.01), whereas BMSCs-exos increased the StAR and CYP11A1 expression ( P < 0.05; Figure 5a – 5c ). We also detected the testosterone concentration in the cell-conditioned medium using ELISA, with similar trends as before, for testosterone concentration in the conditioned medium of each group ( Figure 5d ). However, this ability of BMSCs-exos to improve the testosterone synthesis in CP-exposed TM3 mouse LCs was reversed by 3-MA.

BMSCs-exos regulate AMPK-mTOR signaling pathway in CP-exposed TM3 mouse LCs

Western blot analysis performed on TM3 mouse LCs post CP treatment demonstrated increased p-AMPK protein expression and reduced p-mTOR expression. BMSCs-exos effectively reversed the effect of CP; however, the autophagy inhibitor 3-MA reversed the regulatory effect of BMSCs-exos ( Figure 6 ).

DISCUSSION

While cyclophosphamide is effective in treating many tumors and autoimmune diseases, 35 – 39 it can also cause reproductive side effects in males, 40 – 42 which result in TD. Although TRT has made considerable progress as a therapeutic approach for treating TD, 43 , 44 it has several shortcomings. 45 – 47 In recent years, stem cell-based therapy has received increasing attention; however, its use remains controversial. Concerns include the risk of tumor formation, immune rejection, and ethical issues. 48 Exosomes secreted by stem cells have the therapeutic effect of stem cell transplantation as well as low immunity, no tumorigenicity, and no ethical restrictions. 49 , 50 BMSCs-exos improve CP-induced spermatogenesis dysfunction; however, we did not explore whether they could also improve CP-induced LC dysfunction. Therefore, elucidating the mechanism of BMSCs-exos in improving male reproductive system toxicity caused by CP will provide a vital strategy for the treatment of CP-induced spermatogenesis dysfunction and TD. In this study, we revealed that CP decreased serum testosterone levels in mice. After injections of BMSCs-exos into the tail vein, the testosterone levels partially recovered. In addition, StAR and CYP11A1 mRNA and protein expression levels were upregulated in the CP-exposed TM3 mouse LCs after co-culture with BMSCs-exos. We also observed that the testosterone levels in conditioned cell cultures partially recovered. These results suggest that BMSCs-exos can indeed ameliorate TD induced by CP.

Autophagy is extremely active in LCs, 29 – 31 and can be involved in the regulation of testosterone synthesis in LCs. 33 , 51 This evidence suggests that enhanced autophagy may promote LCs synthesizing testosterone, thereby treating CP-induced TD. However, the effect of BMSCs-exos on autophagy in CP-exposed LCs remains unclear. Our current results suggested that autophagy is inhibited in a CP mouse testicular injury model and CP-exposed TM3 mouse LCs, whereas autophagy levels are increased after BMSC-exo intervention. In addition, BMSCs-exos improved CP-induced low testosterone synthesis by enhancing autophagy in LCs. Furthermore, the ability of BMSCs-exos to treat CP-induced TD was reversed by the autophagy inhibitor 3-MA. These findings suggest that BMSCs-exos can regulate LC autophagy to ameliorate CP-induced TD.

There is crosstalk between exosomes and autophagy in protecting cellular homeostasis; exosomes can regulate the level of autophagy in recipient cells through their transmitted contents. 19 However, the mechanisms regulating autophagy are highly complex and have not yet been fully elucidated. AMPK, an important sensor of intracellular energy molecules, plays a role in many metabolic stresses by downregulating mTOR phosphorylation to achieve energy homeostasis. 52 The AMPK-mTOR signaling pathway participates in the regulation of autophagy. 53 – 56 Liu et al . 57 reported that resveratrol could protect LCs against nicotine-caused oxidative damage by enhancing autophagy via the AMPK-mTOR signaling pathway. Therefore, we further explored whether BMSCs-exos could regulate autophagy in LCs by mediating the AMPK-mTOR signaling pathway, thereby improving CP-induced TD. Our findings suggest that BMSCs-exos ameliorate CP-induced LC dysfunction. This effect promotes p-AMPK protein expression, reduces p-mTOR protein expression, and activates the AMPK-mTOR signaling pathway to promote autophagy in CP-exposed LCs. These results suggest that regulating autophagy in LCs by BMSCs-exos may be achieved by mediating the AMPK-mTOR signaling pathway.

Our study has some limitations that need to be addressed in future studies. First, although we observed that BMSCs-exos could effectively improve CP-induced spermatogenic dysfunction and TD, we did not investigate the specific molecules that are involved in its mechanism of action. Second, while we found that BMSCs-exos could regulate the autophagy of LCs, thereby regulating the synthesis of testosterone by LCs, we did not explore at which stage of autophagy did the BMSCs-exos act to regulate the process. Moreover, we did not investigate how autophagy regulates the synthesis of testosterone by LCs. The therapeutic effect of BMSCs-exos remains to be confirmed by clinical trials.

Future studies need to focus on elucidating the specific molecules that play a therapeutic role in BMSCs-exos, as well as their specific regulatory mechanisms, which will provide a theoretical basis for the application of BMSCs-exos as an effective treatment for CP-induced toxicity in the male reproductive system.

CONCLUSION

Our current results combined with previous research 14 , 34 establish that CP induces cell death in CP-exposed spermatogonia GC1 and TM3 mouse Leydig cells. BMSCs-exos alleviated cell death in CP-exposed spermatogonia GC1 and TM3 mouse Leydig cells and promoted autophagy to improve TD induced by CP by regulating the AMPK-mTOR signaling pathway ( Supplementary Figure 1 ).

AUTHOR CONTRIBUTIONS

QZZ, HYL, and FP designed the study. HYL and FP carried out the animal experiments. HYL, MJP, SLL, and CW carried out the cell experiments. HYL, GYW, XX, and KYX analyzed the experimental data and drafted the manuscript. MKC, JKY, WBG, and CDL participated in revising the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.

Working model of BMSCs-exos in ameliorating CPTD. BMSCs: bone marrow mesenchymal stem cells; BMSCs-exos: BMSCs-derived exosomes; CPTD: cyclophosphamide-induced testosterone deficiency.

ACKNOWLEDGMENTS

This study was supported by grants from the National Natural Science Foundation of China (No. 81772257) and the Third Affiliated Hospital of Southern Medical University President’s Fund (YM2021008).

REFERENCES

1. Zirkin BR, Papadopoulos V. Leydig cells: formation, function, and regulation. Biol Reprod. 2018;99:101–11. doi: 10.1093/biolre/ioy059.

2. Mäkelä JA, Koskenniemi JJ, Virtanen HE, Toppari J. Testis development. Endocr Rev. 2019;40:857–905. doi: 10.1210/er.2018-00140.

3. Zhou R, Wu J, Liu B, Jiang Y, Chen W, et al. The roles and mechanisms of Leydig cells and myoid cells in regulating spermatogenesis. Cell Mol Life Sci. 2019;76:2681–95. doi: 10.1007/s00018-019-03101-9.

4. Emadi A, Jones RJ, Brodsky RA. Cyclophosphamide and cancer: golden anniversary. Nat Rev Clin Oncol. 2009;6:638–47. doi: 10.1038/nrclinonc.2009.146.

5. Ahlmann M, Hempel G. The effect of cyclophosphamide on the immune system: implications for clinical cancer therapy. Cancer Chemotherapy Pharmacol. 2016;78:661–71. doi: 10.1007/s00280-016-3152-1.

6. Al-Homsi AS, Roy TS, Cole K, Feng Y, Duffner U. Post-transplant high-dose cyclophosphamide for the prevention of graft-versus-host disease. Bio Blood Marrow Transplant. 2015;21:604–11. doi: 10.1016/j.bbmt.2014.08.014.

7. Brummaier T, Pohanka E, Studnicka-Benke A, Pieringer H. Using cyclophosphamide in inflammatory rheumatic diseases. Eur J Intern Med. 2013;24:590–6. doi: 10.1016/j.ejim.2013.02.008.

8. Adana MY, Imam A, Bello AA, Sunmonu OE, Alege EP, et al. Oral thymoquinone modulates cyclophosphamide-induced testicular toxicity in adolescent Wistar rats. Andrologia. 2022;54:e14368. doi: 10.1111/and.14368.

9. Nayak G, Rao A, Mullick P, Mutalik S, Kalthur SG, et al. Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis. J Ethnopharmacol. 2020;259:112922. doi: 10.1016/j.jep.2020.112922.

10. Green DM, Liu W, Kutteh WH, Ke RW, Shelton KC, et al. Cumulative alkylating agent exposure and semen parameters in adult survivors of childhood cancer: a report from the St Jude Lifetime Cohort Study. Lancet Oncol. 2014;15:1215–23. doi: 10.1016/S1470-2045(14)70408-5.

11. Chemaitilly W, Liu Q, van Iersel L, Ness KK, Li Z, et al. Leydig cell function in male survivors of childhood cancer: a report from the St Jude lifetime cohort study. J Clin Oncol. 2019;37:3018–31. doi: 10.1200/JCO.19.00738.

12. Salter CA, Mulhall JP. Guideline of guidelines: testosterone therapy for testosterone deficiency. BJU Int. 2019;124:722–9. doi: 10.1111/bju.14899.

13. Barbonetti A, D’Andrea S, Francavilla S. Testosterone replacement therapy. Andrology. 2020;8:1551–66. doi: 10.1111/andr.12774.

14. Fu X, Liu G, Halim A, Ju Y, Luo Q, et al. Mesenchymal stem cell migration and tissue repair. Cells. 2019;8:784. doi: 10.3390/cells8080784.

15. Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther. 2018;9:63. doi: 10.1186/s13287-018-0791-7.

16. Yang M, Lin L, Sha C, Li T, Zhao D, et al. Bone marrow mesenchymal stem cell-derived exosomal miR-144-5p improves rat ovarian function after chemotherapy-induced ovarian failure by targeting PTEN. Lab Invest. 2020;100:342–52. doi: 10.1038/s41374-019-0321-y.

17. Gurung S, Perocheau D, Touramanidou L, Baruteau J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun Signal. 2021;19:47. doi: 10.1186/s12964-021-00730-1.

18. Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature. 2015;527:100–4. doi: 10.1038/nature15376.

19. Yang X, Song X, Li Z, Liu N, Yan Y, et al. Crosstalk between extracellular vesicles and autophagy in cardiovascular pathophysiology. Pharmacol Res. 2021;172:105628. doi: 10.1016/j.phrs.2021.105628.

20. Guo XB, Zhai JW, Xia H, Yang JK, Zhou JH, et al. Protective effect of bone marrow mesenchymal stem cell-derived exosomes against the reproductive toxicity of cyclophosphamide is associated with the p38MAPK/ERK and AKT signaling pathways. Asian J Androl. 2021;23:386–91. doi: 10.4103/aja.aja_98_20.

21. Zhang W, Yang C, Guo W, Guo X, Bian J, et al. [Protective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats. Nan Fang Yi Ke Da Xue Xue Bao. 2018;38:910–6. doi: 10.3969/j.issn.1673-4254.2018.08.02. [Article in Chinese]

22. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147:728–41. doi: 10.1016/j.cell.2011.10.026.

23. Kim KH, Lee MS. Autophagy – a key player in cellular and body metabolism. Nat Rev Endocrinol. 2014;10:322–37. doi: 10.1038/nrendo.2014.35.

24. He Q, Wang L, Zhao R, Yan F, Sha S, et al. Mesenchymal stem cell-derived exosomes exert ameliorative effects in type 2 diabetes by improving hepatic glucose and lipid metabolism via enhancing autophagy. Stem Cell Res Ther. 2020;11:223. doi: 10.1186/s13287-020-01731-6.

25. Jin J, Shi Y, Gong J, Zhao L, Li Y, et al. Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte. Stem Cell Res Ther. 2019;10:95. doi: 10.1186/s13287-019-1177-1.

26. Kuang Y, Zheng X, Zhang L, Ai X, Venkataramani V, et al. Adipose-derived mesenchymal stem cells reduce autophagy in stroke mice by extracellular vesicle transfer of miR-25. J Extracell Vesicles. 2020;10:e12024. doi: 10.1002/jev2.12024.

27. Qu Y, Zhang Q, Cai X, Li F, Ma Z, et al. Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation. J Cell Mol Med. 2017;21:2491–502. doi: 10.1111/jcmm.13170.

28. Wu J, Kuang L, Chen C, Yang J, Zeng WN, et al. miR-100-5p-abundant exosomes derived from infrapatellar fat pad MSCs protect articular cartilage and ameliorate gait abnormalities via inhibition of mTOR in osteoarthritis. Biomaterials. 2019;206:87–100. doi: 10.1016/j.biomaterials.2019.03.022.

29. Yi J, Tang XM. [Lysosomes in the regulation of hormone secretion in endocrine cells. Shi Yan Sheng Wu Xue Bao. 1991;24:203–13. [Article in Chinese]

30. Tang XM. [The autophagic activity of Leydig cells in normal rat testes. Shi Yan Sheng Wu Xue Bao. 1988;21:119–29. [Article in Chinese]

31. Tang XM, Zhang HX. [Cytochemical studies on the formation of multivesicular bodies in Leydig cells. Shi Yan Sheng Wu Xue Bao. 1990;23:453–63. [Article in Chinese]

32. Tang XM, Zhang HX, Yi J. [Leydig cells – a normal cell model of cellular autophagy. Shi Yan Sheng Wu Xue Bao. 1992;25:39–47. [Article in Chinese]

33. Gao F, Li G, Liu C, Gao H, Wang H, et al. Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells. J Cell Biol. 2018;217:2103–19. doi: 10.1083/jcb.201710078.

34. Peng F, Liang HY, Liao SL, Zhai JW, Xue KY, et al. [Bone marrow mesenchymal stem cells-derived exosomes protect TM3 Leydig cells from cyclophosphamide-induced injury. Zhonghua Nan Ke Xue. 2022;28:295–300. [Article in Chinese]

35. Burkitt D. Long-term remissions following one and two-dose chemotherapy for African lymphoma. Cancer. 1967;20:756–9. doi: 10.1002/1097-0142(1967)20:5<756::aid-cncr2820200530>3.0.co;2-p.

36. Mamounas EP, Bryant J, Lembersky B, Fehrenbacher L, Sedlacek SM, et al. Paclitaxel after doxorubicin plus cyclophosphamide as adjuvant chemotherapy for node-positive breast cancer: results from NSABP B-28. J Clin Oncol. 2005;23:3686–96. doi: 10.1200/JCO.2005.10.517.

37. Jayne D, Tyndall A. Autologous stem cell transplantation for systemic lupus erythematosus. Lupus. 2004;13:359–65. doi: 10.1191/0961203304lu1027oa.

38. Tyndall A, Furst DE. Adult stem cell treatment of scleroderma. Curr Opin Rheumatol. 2007;19:604–10. doi: 10.1097/BOR.0b013e3282e6f534.

39. Fassas A, Passweg JR, Anagnostopoulos A, Kazis A, Kozak T, et al. Hematopoietic stem cell transplantation for multiple sclerosis. A retrospective multicenter study. J Neurol. 2002;249:1088–97. doi: 10.1007/s00415-002-0800-7.

40. Trasler JM, Hales BF, Robaire B. A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod. 1987;37:317–26. doi: 10.1095/biolreprod37.2.317.

41. Trasler JM, Hermo L, Robaire B. Morphological changes in the testis and epididymis of rats treated with cyclophosphamide: a quantitative approach. Biol Reprod. 1988;38:463–79. doi: 10.1095/biolreprod38.2.463.

42. Hoorweg-Nijman JJ, Delemarre-van de Waal HA, de Waal FC, Behrendt H. Cyclophosphamide-induced disturbance of gonadotropin secretion manifesting testicular damage. Acta Endocrinol (Copenh) 1992;126:143–8. doi: 10.1530/acta.0.1260143.

43. Brock G, Heiselman D, Maggi M, Kim SW, Rodríguez Vallejo JM, et al. Effect of testosterone solution 2% on testosterone concentration, sex drive and energy in hypogonadal men: results of a placebo controlled study. J Urol. 2016;195:699–705. doi: 10.1016/j.juro.2015.10.083.

44. Kaufman JM, Miller MG, Garwin JL, Fitzpatrick S, McWhirter C, et al. Efficacy and safety study of 1.62% testosterone gel for the treatment of hypogonadal men. J Sex Med. 2011;8:2079–89. doi: 10.1111/j.1743-6109.2011.02265.x.

45. Moss JL, Crosnoe LE, Kim ED. Effect of rejuvenation hormones on spermatogenesis. Fertil Steril. 2013;99:1814–20. doi: 10.1016/j.fertnstert.2013.04.003.

46. Elsherbiny A, Tricomi M, Bhatt D, Dandapantula HK. State-of-the-art: a review of cardiovascular effects of testosterone replacement therapy in adult males. Curr Cardiol Rep. 2017;19:35. doi: 10.1007/s11886-017-0838-x.

47. Jones SD, Dukovac T, Sangkum P, Yafi FA, Hellstrom WJ. Erythrocytosis and polycythemia secondary to testosterone replacement therapy in the aging male. Sex Med Rev. 2015;3:101–12. doi: 10.1002/smrj.43.

48. Ankrum J, Karp JM. Mesenchymal stem cell therapy: two steps forward, one step back. Trends Mol Med. 2010;16:203–9. doi: 10.1016/j.molmed.2010.02.005.

49. Marote A, Teixeira FG, Mendes-Pinheiro B, Salgado AJ. MSCs-derived exosomes: cell-secreted nanovesicles with regenerative potential. Front Pharmacol. 2016;7:231. doi: 10.3389/fphar.2016.00231.

50. Rani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther. 2015;23:812–23. doi: 10.1038/mt.2015.44.

51. Khawar MB, Liu C, Gao F, Gao H, Liu W, et al. Sirt1 regulates testosterone biosynthesis in Leydig cells via modulating autophagy. Protein Cell. 2021;12:67–75. doi: 10.1007/s13238-020-00771-1.

52. Wu H, Song A, Hu W, Dai M. The anti-atherosclerotic effect of paeonol against vascular smooth muscle cell proliferation by up-regulation of autophagy via the AMPK/mTOR signaling pathway. Front Pharmacol. 2017;8:948. doi: 10.3389/fphar.2017.00948.

53. Chun Y, Kim J. AMPK-mTOR signaling and cellular adaptations in hypoxia. Int J Mol Sci. 2021;22:9765. doi: 10.3390/ijms22189765.

54. Alers S, Löffler AS, Wesselborg S, Stork B. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012;32:2–11. doi: 10.1128/MCB.06159-11.

55. Su S, Wang X, Xi X, Zhu L, Chen Q, et al. Phellodendrine promotes autophagy by regulating the AMPK/mTOR pathway and treats ulcerative colitis. J Cell Mol Med. 2021;25:5707–20. doi: 10.1111/jcmm.16587.

56. Chen X, Li C, Chen Y, Ni C, Chen X, et al. Aflatoxin B1 impairs leydig cells through inhibiting AMPK/mTOR-mediated autophagy flux pathway. Chemosphere. 2019;233:261–72. doi: 10.1016/j.chemosphere.2019.05.273.

57. Liu S, Sun Y, Li Z. Resveratrol protects Leydig cells from nicotine-induced oxidative damage through enhanced autophagy. Clin Exp Pharmacol Physiol. 2018;45:573–80. doi: 10.1111/1440-1681.12895.

Associated Data

Supplementary Materials

Working model of BMSCs-exos in ameliorating CPTD. BMSCs: bone marrow mesenchymal stem cells; BMSCs-exos: BMSCs-derived exosomes; CPTD: cyclophosphamide-induced testosterone deficiency.