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. 2021 Apr 26;10(3):436–445. doi: 10.1093/toxres/tfab038

Aristolochic acid I induces impairment in spermatogonial stem cell in rodents

Yongzhen Liu 1,2,#, Xiang He 3,#, Yuli Wang 2, Houzu Zhou 2, Yuan Zhang 2, Jianyun Ma 2, Zhaochu Wang 2, Fangfang Yang 2, Henglei Lu 1,2, Yifu Yang 3, Zhongping Deng 1,✉, Xinming Qi 2,✉, Likun Gong 2,4,✉, Jin Ren 2,✉
PMCID: PMC8201548  PMID: 34141157

Abstract

Aristolochic acid I (AAI) is a natural bioactive substance found in plants from the Aristolochiaceae family and impairs spermatogenesis. However, whether AAI-induced spermatogenesis impairment starts at the early stages of spermatogenesis has not yet been determined. Spermatogonial stem cells (SSCs) are undifferentiated spermatogonia that balance self-renewing and differentiating divisions to maintain spermatogenesis throughout adult life and are the only adult stem cells capable of passing genes onto the next generation. The objective of this study was to investigate whether AAI impairs SSCs during the early stages of spermatogenesis. After AAI treatment, we observed looser, smaller and fewer colonies, decreased cell viability, a decreased relative cell proliferation index, and increased apoptosis in SSCs in a concentration- and/or time-dependent manner. Additionally, AAI promoted apoptosis in SSCs, which was accompanied by upregulation of caspase 3, P53 and BAX expression and downregulation of Bcl-2 expression, and suppressed autophagy, which was accompanied by upregulation of P62 expression and downregulation of ATG5 and LC3B expression, in a concentration-dependent manner. Then we found that AAI impaired spermatogenesis in rats, as identified by degeneration of the seminiferous epithelium, and increased apoptosis of testicular cells. Taken together, our findings demonstrate that AAI causes damage to SSCs and implicate apoptosis and autophagy in this process. The impairment of SSCs may contribute to AAI-induced testicular impairment. Our findings provide crucial information for the human application of botanical products containing trace amounts of AAI.

Keywords: aristolochic acid I, spermatogenesis, spermatogonial stem cells, apoptosis, P53, BAX

Introduction

The mammal testis is composed of seminiferous tubules and interstitial tissues. Seminiferous tubules are the unique site for spermatogenesis, and they include three types of cells: germ cells, Sertoli cells, and myoid cells [1, 2]. Spermatogenesis plays a pivotal role in the continuity of the male germline by producing haploid spermatozoa that fertilize eggs and eventually produce progeny for the next generation [3]. Mammalian spermatogenesis is a highly productive and coordinated process that is subdivided into three successive phases based on functional considerations: the proliferative phase (spermatogonia), the meiotic phase (spermatocytes), and the spermiogenesis phase [4–7]. The life-long maintenance of spermatogenesis is dependent on the biological competence of the extremely rare spermatogonial stem cells (SSCs), which are capable of self-renewal and production of daughter cells to generate terminally differentiated spermatozoa. SSCs are believed to be the least differentiated cell type among spermatogonia, called type A single (As) spermatogonia, which remain as single cells on the basement membrane of the seminiferous tubule [3]. Therefore, drugs/chemicals that disturb spermatogenesis can contribute to testicular injury, while SSC impairment lead to testicular toxicity during the beginning stages of spermatogenesis and ultimately result in permanent male infertility.

Aristolochic acids (AAs) are a family of nitrophenanthrene carboxylic acids found in plants from the Aristolochiaceae family, primarily of the genera Aristolochia and Asarum. Botanical products derived from plants containing AA have been used in traditional folk medicines for thousands of years. AAs have a wide range of pharmacological effects, including antibacterial, anti-inflammatory, analgesic, and anti-tumor effects. AAs are commonly used in the treatment of eczema, pneumonia, and stroke [8]. However, the use of AAs is limited/banned due to toxicity in the liver, kidney, gastrointestinal tract, and carcinogenicity found in recent years [9–18]. Mengs et al. found that spermiogenesis was severely curtailed after acute (single oral or intravenous administration) or subacute (4-week oral administration) AA (a mixture of 77.24% AAI—aristolochic acid I and 21.18% AAII—aristolochic acid II) exposure in rodents at 25 mg/kg or above, whereas cell type impaired was not determined [19, 20]. Apoptosis removes excess, damaged, or infected cells during development and throughout adulthood. It has been reported that germ cells are susceptible to apoptosis due to stress or exposure to exogenous drugs/chemicals [21–28] and excessive apoptosis of germ cells causes infertility [26]. Kwak DH et al. first reported that AAI (the major component of AA mixture) induces testicular Sertoli cell apoptosis through the Akt and ERK1/2 pathway [29]. However, whether AAI also impairs spermatogenesis through direct impairment of germ cells, especially SSC, has not yet been explored.

In this study, we compared the cytotoxicity of AAI in different testicular cell lines and focused on AAI-induced impairment of SSCs in rodents. The effects of AAI on SSCs were determined by evaluating colony morphology, cell proliferation, apoptosis and autophagy in an in vitro SSC mouse model. Then, we used terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and histopathological analysis to demonstrate that AAI impairs germ cells, including SSCs, in rats. Our findings provide crucial information for the human application of botanical products containing trace amounts of AAI.

Materials and Methods

Chemicals and regents

AAI (purity 98%) was purchased from Nanjing Spring & Autumn Biological Engineering Co., Ltd (China). DMSO was purchased from Sigma-Aldrich (USA). StemPro-34 SFM complete culture medium and fetal bovine serum were purchased from Gibico (USA). ViaStain AO/PI Staining Solutions were purchase from Nexcolm (USA). StemPro-34 SFM complete culture medium and fetal bovine serum were purchased from Gibco (USA). ViaStain AO/PI Staining Solutions were purchased from Nexcolm (USA). The Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Dalian Meilun Biotechnology (China). A BCA protein quantitative kit was purchased from Invitrogen (China) and β-Actin antibody was purchased from Abways (China). PageRuler Prestained Protein Ladder was purchased from Applied Biosystem (USA). BAX antibody was purchased from Shanghai Univ Bio-Technology Co., Ltd (China). P53 antibody was purchased from Abclonal (USA). Phospho-p53 (Ser15), P62, and Bcl-2 antibodies were purchased from Abcam (USA). ATG5 antibody was purchased from Proteintech (USA), and LC3 antibody was purchased from CST (USA).

S‌SC isolation and culture conditions

Testicular cells were isolated from 6-day-old male BDF1 mice by two-step enzymatic digestion [30, 31]. Briefly, testis cells were digested with collagenase/DNase solution for 20 min, followed by dispase/DNase solution digestion for 15 min. Dissociated testis cells were seeded onto 0.1% (w/v) gelatin-coated tissue culture plates for differential adhesion to enrich the SSCs. Enriched SSCs were then transferred to mitomycin C inactivated mouse embryonic fibroblast (iMEF)-coated dishes for culture in StemPro-34 SFM Complete Medium supplemented with 25 mg/ml of insulin, 100 mg/ml of transferrin, 60-mM putrescine, 30-nM sodium selenite, 6 mg/ml of d-(+)-glucose, 30 mg/ml of pyruvic acid, 1 ul/ml of DL-lactic acid, 5 mg/ml of bovine albumin, 2-mM l-glutamine, 5 × 10−5 M 2-mercaptoethanol, minimal essential medium (MEM) vitamin solution, MEM nonessential amino acid solution, 10−4 M ascorbic acid, 10 mg/ml of d-biotin, 30 ng/ml of b-estradiol, 60 ng/ml of progesterone, 20 ng/ml of mouse epidermal growth factor, 10 ng/ml of human basic fibroblast growth factor, 103 U/ml of ESGRO (mouse leukemia inhibitory factor), 10 ng/ml of recombinant rat glial cell line-derived neurotrophic factor, and 1% fetal bovine serum. The cells were maintained at 37°C in an atmosphere of 5% carbon dioxide in air.

S‌SC characterization by immunofluorescence staining and confocal microscopy

The morphology of SSCs cultured on iMEFs was monitored daily under an inverted phase contrast microscope (Leica, Germany). For immunofluorescence analysis, 5 days after seeding, SSCs were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were then incubated with PBS supplemented with 0.1% Triton-X (Sigma, USA) for 15 min followed by overnight incubation at 4°C with the following primary antibodies: anti-MILI antibody (1:200; CST, USA) and anti-OCT4 antibody (1:400; Abcam, UK). The cells were then incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 594 goat anti-rabbit, 1:800; Invitrogen, USA) at 37°C for 1 h. Then, DAPI was added for nuclear staining, and the cells were observed by confocal microscopy (Leica TCS-SP8, Germany).

Colony morphological observation and cytotoxicity assessment

SSCs were seeded onto iMEF-coated 24-well plates at a density of 1.5 × 105 cells/well. GC-1 (mouse spermatogonia-like cells, NCACC, China), TM4 (mouse Sertoli cells, ATCC, USA), and GC-2 (mouse spermatocyte-like cells, ATCC, USA) cells were seeded in 12-well plates at densities of 1.2 and 0.9 × 104 cells/well. The culture medium for SSC, TM4, and GC-2 cells was StemPro-34 SFM (Invitrogen) as described above, DMEM/F12 medium (Gibco, USA) supplemented with 5% FBS and 2.5% horse serum, and DMEM (Gibco, USA) supplemented with 10% FBS, respectively. The AAI concentrations were 0, 0.7825, 1.5625, 3.125, 6.25, 9.375, 12.5, 18.75, 25, 50, and 100 μM for the treatment of SSCs; 0, 0.7825, 1.5625, 3.125, 6.25, 9.375, 12.5, 18.75, 25, 50, and 100 μM for TM4 cells; and 0, 0.7825, 1.5625, 2.00, 3.125, 4.00, 6.25, 9.375, 12.5, 18.75, and 25 μM for GC-1 cells and 0, 3.125, 6.25, 9.375, 12.5, 15, 18.75, 25, 37.5, 50, and 100 μM for GC-2 cells. The cells were exposed to AAI 24 h after seeding, and the morphology was observed under an inverted phase contrast microscope 24, 48, and 72 h after AAI treatment. After 72 h of treatment, the cells were harvested by 0.05% or 0.25% trypsin/EDTA digestion, centrifuged, and resuspended in the respective culture medium (for SSCs, 1 h of differential adhesion was performed, and the floating cells were collected). The cells were then stained with AO/PI and counted using a dual fluorescence automatic Cellometer K2 Cell Viability Counter (Nexcelom, USA). A half-inhibitory concentration (IC50) was calculated. The controls were normalized to 100% for each assay, and the treatment results were expressed as the percentage of the controls.

Flow cytometry assays to detect SSC apoptosis

Annexin V-FITC staining was used to quantify the percentage of cells that were actively undergoing apoptosis. Annexin V-FITC staining relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. Annexin V-FITC staining precedes the loss of membrane integrity, which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V-FITC is typically used in conjunction with a vital dye such as propidium iodide (PI) to identify early apoptotic cells (PI negative, Annexin V-FITC positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. For example, cells that are considered viable are Annexin V-FITC and PI negative; cells that are in early stages of apoptosis are Annexin V-FITC positive and PI negative; and cells that are in the late stages of apoptosis or already dead are both Annexin V-FITC and PI positive.

An Annexin V-FITC apoptosis detection kit (Meilun Bio., Dalian, China) was used in this study to detect apoptosis. SSCs were seeded in 12-well plates at a density of 3 × 105 cells per well, and after 24 h of incubation, the cells were treated with AAI at 0, 6.25 and 12.5-μM concentrations for 24, 48, or 72 h, respectively. After treatment, both adherent and floating cells were collected and counted using a dual fluorescence automatic Cellometer K2 Cell Viability Counter (Nexcelom, USA). Next 1.0 × 106 cells were suspended in 100 μl of Annexin V binding buffer and stained with 5 μl of Annexin V-FITC conjugate and 10 μl of PI solution. The cells were then incubated for 15 min at 37°C in the dark. The stained cells were subsequently resuspended in 400 μl of Annexin V binding buffer and analyzed by flow cytometry (BD, USA) within 1 h. The following controls were included: (i) unstained cells; (ii) cells stained with Annexin V-FITC (no PI); and (iii) cells stained with PI (no Annexin V-FITC). Cells were incubated in a 60°C water bath for 3 min to induce death.

Protein extraction and western blotting

The SSCs were plated in 6-well plates at a density of 6 × 105 cells per well and treated with 0, 6.25, and 12.5-μM AAI for 72 h. Then, the SSCs were lysed in cell lysis buffer, and the protein concentrations were determined using the BCA assay (Invitrogen, Shanghai, China). A total of 10 μl of protein sample was separated by 12% SDS-PAGE and transferred onto PVDF membranes. Blots were blocked with 5% milk in TBS + 0.05% Tween 20 (TBST), incubated at room temperature for 1 h, and then incubated with primary antibodies overnight at 4°C. Blots were then washed in TBST and incubated for 1 h with secondary polyclonal antibodies. Blots were then washed in TBST and developed using ECL chemiluminescence (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland).

Animal studies

Animal protocol was reviewed and approved by the Shanghai Institute of Materia Medica Animal Care and Use Committees (IACUC No. 2018-01-RJ-156). Six-week-old male Sprague–Dawley (SD) rats were randomly divided into 4 groups (10 rats/group) after acclimatization for 5–7 days. AAI (0, 0.1, 1, or 10 mg/kg) was administered to SD rats for up to 26 weeks [once a day, 5 days a week (Monday–Friday)], and the rats were sacrificed by the end of Week 26 for collection of the testes. Mengs and Stotham [20] reported that male Wistar rats exposed daily to AAs (a mixture of 77.24% AAI and 21.18% AAII) by gavage at 1, 5, or 25 mg/kg for 4 weeks developed mild testicular degeneration at 5 mg/kg and severe degeneration at 25 mg/kg. Therefore, a high dose of 10 mg/kg was chosen considering the longer dosing duration used in the present study. According to the Ch. P 2015 Edition [32], the maximal recommended human daily dose (MRHD) for AAI is 0.6 mg, which is equivalent to 0.06 mg kg−1 d−1 in rats, based on the body surface area. Thus, the doses of 0.1, 1, and 10 mg/kg used in present study were approximately 1.67-, 16.7-, and 167-fold the MRHD for AAI, respectively. Investigating the testicular toxicity at much wider dose ranges will provide additional information for further use of botanical products containing trace amounts of AAs in human.

Testicular tissues from all animals were collected at necropsy, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (3 μm), and stained with hematoxylin and eosin (H&E) for histological evaluation. Subsequently, 5 μm sections were used for the TUNEL assay using an in situ Apoptosis Detection Kit (Meilunbio, China) according to the instructions.

Statistical analysis

Statistical analysis of the results was performed using GraphPad Prism 8.1.1 (GraphPad Prism Software Inc., San Diego, CA). In all experiments, the mean value of three separate experiments performed ± standard error of the mean (SE) were calculated. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test. P < 0.05 was considered statistically significant.

Results

Isolation and identification of SSCs

SSCs isolated from 6-day-old mice were cultured and identified for further in vitro studies. The morphology of SSCs was observed under an inverted phase contrast microscope, and as expected, SSCs proliferated by forming typical and compact cobble-like or grapelike colonies (Fig. 1A and B), the same morphology as reported previously [31].

Figure 1.

Figure 1

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Morphology of SSC colonies and immunofluorescence analysis of MILI and OCT4. (A, B) The appearance of SSC colonies (light field/phase contrast). scale bar: 100 μm. (C) Immunofluorescence analysis revealed positive MILI staining in the cytoplasm. (D) Immunofluorescence analysis revealed positive OCT4 staining in the nucleus.

MILI, a PIWI protein, and OCT4, one of the stem cell markers, both play important roles in spermatogenesis and stem cell self-renewal and maintenance [33, 34]. MILI is exclusively expressed in germ cells, including SSCs and is concentrated in perinuclear cytoplasmic granules in the developing testis [35, 36]. OCT4 is expressed in nuclei of undifferentiated spermatogonia, including SSCs, at prepubertal stages [34]. Thus, MILI and OCT4 staining was assessed in SSCs herein. The results demonstrated positive staining of MILI and OCT4, and MILI was distributed in the perinuclear cytoplasm, while OCT4 was mainly distributed in the nucleus (Fig. 1C and D).

A‌AI induces cytotoxicity in different testicular cells

It has been reported that AAI induces testicular Sertoli cell (TM4) apoptosis in testicular somatic cells [29]. To investigate whether AAI impairs spermatogenesis directly through germ cells, we compared the cytotoxicity of AAI in four different testicular cell lines. We found that the viability and relative proliferation index decreased in all 4 cell lines in a dose-dependent manner 72 h after AAI treatment (Fig. 2A). The IC50 (95% CI) values were 8.710 μM (7.013–10.74 μM), 1.728 μM (1.519–1.917 μM), 19.53 μM (16.30–23.24 μM), and 15.52 μM (13.09–18.28 μM) in SSC, GC-1, GC-2, and TM4 cells, respectively (Fig. 2A and B). These findings indicated that, in addition to Sertoli cells, germ cells, including SSCs, are all impaired during AAI exposure.

Figure 2.

Figure 2

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Cytotoxicity comparison of different testicular cell lines and colony morphology of SSCs after AAI treatment. (A) Relative cell proliferation inhibition curve of different testicular cell lines after AAI treatment for 72 h. (B) IC50 values and 95% CI in different testicular cell lines after AAI treatment for 72 h. (C) The appearance of SSC colonies after 72 h of AAI treatment is presented at representative concentrations. Scale bar: 200 μm.

A‌AI induces changes in the SSC colony morphology

SSCs are the only stem cells in adults that can transmit genetic information to the next generation and maintain life-long spermatogenesis. Any toxicity induced by a chemical to SSCs will have an impact on spermatogenesis and finally lead to male infertility. Thus, we investigated SSC impairments after AAI treatment. In this study, we monitored the morphological changes of SSCs under an inverted phase contrast microscope during AAI treatment. SSCs were treated with AAI at concentrations ranging from 0.7825 to 100 μM for 72 h, and typical cobble-like or grapelike colonies were observed with 0-μM AAI treatment (Fig. 2C). The morphology of the SSC colonies was not changed after 24 h of AAI treatment. However, looser, smaller and fewer colonies were observed in a dose- and time-dependent manner beyond 48 h of treatment. After 72 h of treatment, SSC colonies were rarely observed, but feeder cells were present at the 25-μM AAI concentration and above (Fig. 2C).

A‌AI induces cell proliferation inhibition, apoptosis and autophagy in SSCs

AO/PI assays indicated that AAI decreased the number and viability of SSCs in a dose- and time-dependent manner (Fig. 2). AAI has been reported to cause nephropathy first by inducing renal tubular cell autophagy and apoptosis [37]. To investigate whether autophagy and apoptosis are also involved in AAI-induced impairment of SSCs, first, a flow cytometry assay was used to detect the apoptotic effects on SSCs after AAI treatment; next, western blotting was performed to investigate whether there were any effects on the expression of autophagy- and apoptosis-associated proteins. SSCs were treated with 0, 6.25, and 12.5-μM AAI for 72 h. Flow cytometry assays indicated that decreased cell viability (relative proliferation index) accompanied by increased apoptosis was observed in a time- and dose-dependent manner after AAI treatment (Fig. 3A–C). Western blotting results demonstrated increased expression of the apoptosis-associated proteins caspase 3, P53, and BAX and decreased expression of Bcl-2 in a dose-dependent manner (Fig. 3D and E). Concomitantly, the autophagy-related proteins ATG5 and LC3B decreased, and P62 increased markedly in a dose-dependent manner (Fig. 3D–G).

Figure 3.

Figure 3

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AAI-induced apoptosis and autophagy of SSCs. (A, B) Mean values ± SE (N = 3) of the relative proliferation index (%) and apoptosis (%) are presented. *P < 0.05, and ***P < 0.001 vs control group. (C) Representative scatter plots demonstrate a tested cell population divided by a gate into four subpopulations: viable cells are Annexin V-FITC and PI negative; early apoptotic cells are Annexin V-FITC positive and PI negative; and late apoptotic or dead cells are both Annexin V-FITC and PI positive. (D–F) western blotting demonstrated that AAI-induced apoptosis and autophagy 72 h after AAI treatment (N = 3). Mean values ± SE (N = 3) of the relative proliferation index (%) and apoptosis (%) are presented. *P < 0.05, *P < 0.01, and ***P < 0.001 vs control group.

A‌AI impairs spermatogenesis and induced testicular cell apoptosis in SD rats

To investigate whether AAI also impairs germ cells, including SSCs, in vivo, AAI (0, 0.1, 1 or 10 mg/kg) was administered to SD rats for 26 weeks, and the rats were sacrificed by the end of Week 26. Histopathological examination demonstrated slight to moderate degeneration of the seminiferous epithelium, characterized by the presence of partial apoptotic or necrotic testicular cells and, occasionally, the presence of multinucleated giant cells and vacuolation of germ/Sertoli cells at 10 mg/kg. The affected cells were mainly distributed in the 1–2 layers of cells at the area of the basement membrane of the seminiferous tubules (Fig. 4A). No histopathological changes were observed at 1 and 0.1-mg/kg doses of AAI (Fig. 4A). Similarly, the TUNEL assay indicated that the apoptotic cells were mainly distributed at/near the area of the basement membrane where spermatogonia, including SSCs, primary spermatocytes and Sertoli cells, were located, and fewer were distributed near the lumen side of the seminiferous tubules where spermatids were located (Fig. 4B). These findings indicate that germ cells are also impaired after AAI treatment in rats.

Figure 4.

Figure 4

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AAI-induced testicular injury in Sprague–Dawley (SD) rats. H&E staining and TUNEL assays demonstrated that AAI-induced testicular injury after 26 weeks of AAI oral administration (once a day, 5 days a week (Monday to Friday)) at 10 mg/kg. The ×40 images of the seminiferous tubules are the enlarged views of tubules indicated by triangles in the ×10 images at respective dose levels. (A) H&E staining was performed. Arrows illustrate necrotic or apoptotic germ cells; arrowheads illustrate vacuolation in the cytoplasm of testicular cells. Scale bar: ×10 = 200 μm; ×40 = 50 μm. (B) TUNEL assays demonstrated apoptosis in Sprague–Dawley rats after oral administration of AAI at 10 mg/kg for 26 weeks. Scale bar: ×10 = 100 μm; ×40 = 25 μm.

Discussion

Male infertility, becoming a serious social problem worldwide, is mainly related to spermatogenesis malfunctions that are caused by known or unknown factors and finally result in azoospermia, oligozoospermia, or asthenospermia. The life-long maintenance of spermatogenesis is dependent on the biological competence of the extremely rare SSCs, which are capable of self-renewal and production of daughter cells to generate terminally differentiated cells, spermatozoa. Thus, SSC impairment induce testicular toxicity during early stages of spermatogenesis and ultimately result in permanent male infertility, which can only be treated by SSC transplantation. Because it is neither practical nor feasible to conduct clinical studies assessing male fertility by pregnancy rate, the main outcome measures of clinical studies rely on semen parameters. Thus, a thorough evaluation of the adverse effects of a drug on testes in humans is challenging [38]. In compliance with the 3R principles, it is valuable and important to use in vitro models for rapid screening of candidate drugs that will not cause reproductive toxicity. It is also important to assess the effects of candidate drugs at the SSC stage during drug development, to investigate the toxic mechanisms of chemicals with testicular toxicity, and to find solutions to treat infertility.

Autophagy and apoptosis are two evolutionarily conserved processes that regulate cell fate in response to cytotoxic stress. The balance between autophagy and apoptosis is regulated by various cell signals, and crosstalk between these pathways determines cell fate during stress. Autophagy is a highly conserved catabolic pathway that degrades cellular macromolecules and organelles and plays critical roles in tissue development, differentiation, and homoeostasis [39]. P53 has been accepted as a bimodal regulator of autophagy: nuclear p53 acts as a transcription factor that induces autophagy by upregulating target genes, whereas cytoplasmic p53 suppresses autophagy post-transcriptionally [40–45]. Apoptosis plays an important role in regulating spermatogenesis in various mammalian species including humans [46]. Germ cells are susceptible to apoptosis due to stress or exposure to exogenous drugs/chemicals [26]. Relatively high rates of apoptosis have been reported in testicular biopsies from infertile men with different degrees of testicular insufficiency [47]. Tumor suppressor P53 promote the expression of apoptotic proteins as a transcription factor and also directly activates Bax, a Bcl-2 family proapoptotic protein, in a transcription-independent manner, thus causing apoptosis [48–57]. P53 is reported to play an important role in modulating apoptosis and spermatogenesis in various mammalian species including human [58–60]. The Bax gene is a P53 target and is up-regulated during p53-mediated apoptosis [57]. Experiments performed in Bax-deficient mice strongly support that Bax is a primary target for p53-induced apoptosis in a variety of cells. It is also reported that proapoptotic BAX appears to be essential for progression through the first wave of spermatogenesis [61].

AAI is a known cytotoxic and nephrotoxic carcinogen. It has been reported that the acute phase of AAI-induced nephropathy is associated with the induction of ATG5-dependent autophagy, which promotes renal tubular cell apoptosis [37]. AAI has also been reported to impair spermatogenesis in rodents [19, 20, 62]. However, the cell types impaired during spermatogenesis remains poorly understood, and whether autophagy and apoptosis are involved in the impairment in the early stages of spermatogenesis (e.g. SSC stage) has not yet been determined. Cytotoxicity assays demonstrated that in addition to Sertoli cells, germ cells, including SSCs, are all impaired during AAI exposure. Because spermatogonia and spermatocytes are daughter cells of SSCs, any injury to SSCs might ultimately affect the daughter cells; therefore, we investigated the effects of AAI on SSCs. We found that AAI-induced alteration of colony morphology, inhibited proliferation and induced apoptosis in SSCs. In addition, we found that AAI promoted apoptosis of mouse SSCs, which was accompanied by upregulation of P53, BAX, and Caspase 3 expression and downregulation of Bcl-2 expression, and suppressed autophagy, which was accompanied by upregulation of P62 expression and downregulation of ATG5 and LC3B expression. These findings indicated that both apoptosis and autophagy might contribute to the impairment of SSCs after AAI exposure and that the mechanism is somehow different from AAI-induced injury in renal tubular cells [37]. However, the role and mechanism of apoptosis and autophagy in AAI-induced SSC impairment still needs to be clarified in the future. In this study, we further demonstrated that AAI impaired other types of germ cells, including spermatogonia and spermatocytes, in rats. Our in vivo findings in rodents were consistent with in vitro results. Taken together, AAI-induced SSC impairment, and this early-stage SSC impairment, may contribute to AAI-induced impairment of the testes.

Until the 2020 edition of the Ch. P, only one botanical product containing trace amounts of AAs, namely, Asarum heterotropoides/Xixin, was included in the Ch. P, and an MRHD of 3 g with a maximal content of AAI not exceeding 0.001% is recommended [63]. Accordingly, the MRHD of AAI in humans is 0.03 mg, which is equivalent to 0.003 mg kg−1 d−1 in rats based on the surface area of the body. The high dose that caused testicular toxicity in the 26-week rat study was 10 mg/kg, which is approximately 3333-fold the MRHD of AAI in the Ch. P 2020 Edition. No testicular toxicity was observed at or below 1 mg/kg of AAI, which is at least 333-fold the MRHD. Our results indicated that there is enough safety margin on AAI-induced testicular toxicity for the application of the botanical products containing trace amounts of AAI in human under Ch. P 2020 recommendations. Our results might partially explain why testicular impairment has rarely been reported, while toxicity in the liver, kidney, gastrointestinal tract, and carcinogenicity of AAI has been widely reported.

Conclusion

Findings from our studies suggested that AAI could induce SSC impairment at the early stage of spermatogenesis, which may contribute to testicular impairment after AAI exposure, and autophagy and apoptosis were involved in this process. Our findings provide crucial information for human application of botanical products containing trace amounts of AAI.

Acknowledgments

We would like to thank Professor Jing-song Li’s Laboratory (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) for gifting us the BDF1 mice for SSC separation and enrichment, and for providing training on SSC culture.

Funding

This work was supported by the National Science and Technology Major Project (2018ZX09201017-004).

Conflicts of Interest

There are no conflicts of interest to declare.

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