Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol

Abstract

Background: Baked carbohydrate-rich foods are the main source of acrylamide (AA) in the general population and are widely consumed by teenagers. Considering the crucial development of the reproductive system during puberty, the health risks posed by AA in adolescent males have raised public concern.

Methods: In this study, we exposed 3-week-old male pubertal mice to AA for 4 weeks to evaluate its effect on spermatogenesis using computer-assisted sperm analysis (CASA) and historical analysis. Flow cytometric analysis and meiocyte spreading assay were conducted to assess meiosis in mice. The expression of meiosis-related proteins and double-strand break (DSB) proteins were evaluated by immunoblot analyses. Additionally, isolated spermatocytes were used to explore the role of resveratrol in AA-induced damages of meiosis.

Results: Our results showed that AA decreased the testicular and epididymal indexes, reduced sperm count and motility, and induced morphological disruption of the testes in pubertal mice. Subsequent meiotic analysis revealed that AA increased the proportion of 4C spermatocytes and decreased the proportion of 1C spermatids. The expression levels of meiosis-related proteins (SYCP3, Cyclin A1 and CDK2) were downregulated, and signaling proteins (γH2AX, p-CHK2 and p-ATM) expression levels were upregulated in AA-treated mice testes. Similar expression patterns were observed in primary spermatocytes treated with AA and these effects were reversed significantly by resveratrol.

Conclusions: Our results indicate that AA induces meiotic arrest via persistent activation of DSBs, which may contribute to AA-compromised spermatogenesis. Resveratrol could serve as a potential therapeutic agent against AA-induced meiotic toxicity. These data highlight the importance of natural product supplementation for treating AA-related reproductive toxicity.

Keywords

Acrylamide spermatocytes meiosis meiotic DNA strand breaks resveratrol

Introduction

Acrylamide (AA) is a surprisingly common pollutant. It is extensively used in polymeric and copolymeric products, including paper, dyes, and cosmetics. Furthermore, AA is formed through a thermal process during the Maillard reaction of carbohydrate-rich foods or tobaccos.^1,2 As a hydrophilic and low molecular weight chemical, AA can be absorbed via ingestion, inhalation, and dermal contact and is subsequently metabolically activated by cytochrome P450 2E1 (CYP2E1) to form glycidamide (GA); GA is recognized as an activated metabolite with higher carcinogenic activity than that of AA. This carcinogenic activity is due to its covalent interaction with DNA strands.^3,4 The unavoidable presence of AA in our daily life has gained increasing public health attention and numerous studies have been performed to explore the deleterious effects of AA on different body systems. These include the male reproductive system and spermatogenesis, a tightly and precisely coordinated process that is vulnerable to environmental influences.^5,6 Acrylamide has been shown to inhibit the production of the sex hormone testosterone in the testis, and Leydig cells of adult mice, by triggering the phosphorylation of ERK1/2.⁷ Paternal AA exposure has been associated with transgenerational adverse effects on sperm parameters such as sperm concentration, motility and malformation in mice.⁸ However, these studies were performed to mimic human exposure to AA in sexually mature adults and not adolescents. Adolescents are more likely than adults to consume AA-contaminated baked and fried food such as potato crisps, crackers and coffee.^9,10 Moreover, since adolescents are going through pubertal reproductive development, they are more vulnerable than adults to reproductive toxicant exposure,¹¹ though the evidence to support this quite limited.

Meiosis is an essential stage of specialized cell division in the production of haploid gametes, characterized by DNA replication, homologous chromosome pairing, double-strand break (DSB) repair, and chromosome segregation.¹² DNA damage is a common biological event of meiosis that induces chromatin remodeling and synaptonemal complex disassembly.¹³ Persistent meiotic DSBs that cannot be accurately repaired may lead to the impaired transmission of intact genetic information to subsequent generations and ultimately to meiotic arrest.¹⁴ Considering the genotoxicity of AA and its metabolite GA, the hypothesis that disrupted meiotic progression may be a potential way in which AA induces DNA damage and compromises spermatogenesis in the testis seems reasonable. A recent study showed that pubertal exposure to AA was associated with altered ratios of leptotene, zygotene, pachytene and diplotene spermatocytes and decreased expression of the meiosis-associated protein SYCP3, suggesting an interference with meiotic progression triggered by AA.¹⁵ The underlying mechanisms by which AA induces meiotic disruption remains unclear. There is little evidence available to support any potential therapeutic agents that may protect individuals against AA related-reproductive toxicity, especially meiotic toxicity.

Resveratrol (3,4,5-trihydroxystilbene) is a well-known natural stilbene compound present in grapes, legumes and mulberries.¹⁶ It is widely used as potential therapeutic agent due to its various beneficial activities, such as potent antioxidant and anti-inflammatory properties, cardioprotective and neuroprotective activities, and powerful chemopreventive effects.^17–19 It also plays a key role in DNA repair and cellular detoxification.²⁰ Previous studies have demonstrated that resveratrol alleviates the effect of xenobiotic (such as irradiation, fungicide and ethanol)-induced DNA damage in vivo and in vitro.^21–23 CYP2E1 enzymatic activity for is essential for AA-triggered DNA damage and resveratrol has been reported to downregulate the expression of CYP2E1 and inhibit its enzymatic activity.²⁴ Therefore, resveratrol could serve as a potential therapeutic agent for AA-induced meiotic dysfunction.

In this study, in vivo experiments were performed to determine the possible deleterious effects of AA on spermatogenesis, especially meiosis progression, and to explore the underlying mechanisms behind these deleterious effects. Primary spermatocytes were isolated and used to investigate the protective effects of resveratrol on AA-induced DNA damage and meiotic dysfunction. These results reveal the toxicological importance of meiosis in AA-related male reproductive disorders during adolescence and suggest a possible approach for treating AA-induced meiotic disruption.

Method

Chemicals and reagents

Acrylamide (A9099, purity ≥99%), collagenase IV (C4-BIOC), trypsin (T4424), DNase I (11284932001), propidium iodide (PI, 537060), Triton X-100 (T8787) and RNase A (70856) were obtained from Sigma‒Aldrich (St Louis, MO, USA). Dulbecco’s modified eagle’s medium (DMEM), M199 medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Resveratrol (HY-16561) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Streptomycin, penicillin, and cell counting kit-8 (CCK-8) reagents were purchased from Beyotime (Shanghai, China). Antibodies specific for PCNA (ab92552), MVH (ab270534) and γH2AX (ab303656) were obtained from Abcam (Cambridge, MA, USA). Antibody against SYCP3 (23024-1-AP) was purchased from Proteintech (USA). Antibodies against GAPDH (AF7021), Cyclin A1 (AF5313), p-ATM (AF8225), and p-CHK2 (AF3036) were obtained from Affinity (China). CDK2 (18048) was purchased from Cell Signaling Technology (Beverly, MA, USA).

Experimental animals and study design

Twenty pubertal male C57BL/6 mice (3 weeks old, 15–18 g, Animal Core Facility of Nanjing Medical University, Nanjing, China) were housed in a standard environment using a controlled temperature (25 ± 1°C), relative humidity (50–60%) and light/dark cycle (12 h/12 h). All animals had free access to standard rodent food and water. The mice acclimatized for 1 week were randomly divided into two groups (10 mice per group, 5 mice per cage). In the control group, mice were exposed to vehicle solution (formulated by mixing 3 mL saline and 147 mL drinking water), while in the AA group, mice were administered 200 mg/L AA [formulated by mixing 3 mL AA solution (10 mg/mL) and 147 mL drinking water]. The mixed solutions in both groups were renewed every 3 days. The AA concentration was chosen based on the human AA exposure levels reported in previous studies.^25,26 After a 4-week treatment, the mice were euthanized by cervical dislocation, and the testis and epididymis were isolated immediately for subsequent analyses. All the experimental protocols in this study were approved by the Animal Investigation Ethics Committee of Nanjing Medical University (2003001-1) and followed the institutional and national animal welfare guidelines.

Computer-assisted sperm analysis

Sperm parameters were measured according to a previous report.²⁷ Briefly, at the experimental endpoint, the freshly dissected cauda epididymis (N = 10 mice for each group) were washed three times with warm (37°C) phosphate-buffered saline (PBS) and then excised using a surgical blade. Spermatozoa were released into the M199 medium and incubated for 5 min at 37°C. Sperm counts and motility were further evaluated using a Computer-assisted sperm analysis (CASA) system (Hamilton Thorne, MA, USA): sperm samples (20 μL) were placed on glass slides, heated to 37°C, and images were immediately recorded and digitized at ×400 magnification. Ten images were captured for each sample, and the results were analyzed using OpenCASA software.

Histological observation of the testes and epididymides

At the experimental endpoint, freshly isolated testes and epididymides (N = 5 mice per group) were rinsed three times with PBS and fixed with Davidson’s fixative solution for at least 24 h. The fixed testes and epididymides were then routinely dehydrated with graded ethanol, embedded in paraffin, serially sliced (5 μm) and stained with hematoxylin and eosin (H&E). A Panoramic MIDI scanner (3DHISTECH, Budapest, Hungary) was employed for histological observation of the testes and epididymides.

Flow cytometric analysis

At the experimental endpoint, testicular tissues (N = 3 mice per group) were routinely decapsulated, rinsed three times with PBS and mechanically minced into pieces. The testicular pieces were then subjected to two-step collagenase enzymatic digestion; testicular pieces were incubated with collagenase IV (0.75 mg/mL) for 30 min at 37°C and trypsin solution (1 mg/mL with 5 μg/mL DNase Ⅰ) for 5 min at 37°C. The digested testicular pieces were then filtered through a nylon mesh (100 μm) to obtain a single-cell suspension. The filtered spermatogenic cells were fixed with 75% ethanol and stored at 4°C. The fixed cells were incubated with a mixture of PBS containing PI (50 μg/mL), Triton X-100 (0.1%) and RNase-A (500 μg/mL) for 30 min in the dark. The percentages of the 1C, 2C and 4C-DNA cell populations were determined using a FACS Calibur flow cytometry (BD Biosciences, San Jose, CA, USA).

Meiocyte spreading assay

A drying-down technique was used to perform meiotic spread assays as previously reported.¹⁴ In brief, at the experimental endpoint, testes tissues (N = 3 mice for each group) were decapsulated and incubated with hypotonic extraction buffer for 60 min on ice. One-inch seminiferous tubules were incubated with sucrose solution and spread onto two slides with a fixative solution. The prepared slides were dried for 2−3 h before being analyzed by immunofluorescence.

Cell isolations, cultures, and treatments

Primary spermatocytes were isolated from the testis of male C57BL/6 mice (17 days postpartum, Animal Core Facility of Nanjing Medical University, Nanjing, China) according to the previously described STA-PUT velocity sedimentation method.²⁸ In brief, a single-cell suspension was prepared in accordance with the previous flow cytometric analysis. The obtained spermatogenic cells were then loaded onto a 2−4% BSA gradient in DMEM for at least 3 h at 4°C to separate different types of spermatogenic cells based on their density. Cell fractions (10 mL/pool) were collected in numbered tubes and identified by phase contrast microscopy (JOEL, Tokyo, Japan). Pachytene spermatocytes with purity ≥60% were mainly observed in fractions of approximately 10–15 and were used for subsequent analyses.

The newly isolated spermatocytes were cultured in DMEM supplemented with 5% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL) and maintained in a humidified incubator with 5% CO₂ at 34°C. The cells were seeded on 96-well plates at a density of 0.5 × 10⁶ cells/cm² overnight and then treated with a series of concentrations of AA (10 μM–2500 μM) for 24 h to screen for the nonobserved cytotoxic level using CCK-8 reagents. Resveratrol was used at a concentration of 50μM (dissolved in DMSO), based on the optimal concentrations reported by previous studies.^29,30

Immunoblot analyses

At the experimental endpoint, testicular tissues (N = 5 mice per group) and primary spermatocytes were homogenized in lysis buffer for 30 min on ice. After centrifugation (12000×g) for 30 min at 4°C, samples were denatured and subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis for 1.5 h. Proteins on the gel were then transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were incubated with specific antibodies against PCNA, MVH, SYCP3, CDK2, CyclinA1, γH2AX, p-ATM or p-CHK2 overnight at 4°C. The blots were detected using a BeyoECL Star Immunoblotting Assay kit (Beyotime, Shanghai, China). GAPDH was used as an internal control and ImageJ software was used to quantitatively evaluate the protein bands on the membranes.

Immunofluorescence analyses

At the experimental endpoint, testicular tissues (N = 3 mice per group) were isolated, washed, and immersed in Tissue-Tek OCT (Sakura, Tokyo, Japan). Cryosections (10 μm) were obtained using a freezing microtome (Thermo, MA, USA), and were routinely fixed with a mixed solution of methanol and acetone (1:1 ratio). Samples were then blocked with 0.1 M PBS supplemented with 5% BSA and 0.1% Triton X-100, and incubated with specific primary antibodies against SYCP3 overnight at 4°C. For the meiocyte spreading assay, the previously prepared slides were washed with dilution buffer and incubated with anti-rabbit-SYCP3 and anti-mouse-γH2AX overnight at 4°C. The next day, the slides were incubated with the corresponding secondary antibodies for 1 h at room temperature. Cell nuclei were stained with DAPI for 15 min at 37°C. Confocal fluorescence microscopy (Carl Zeiss, Jena, German) was used to capture and analyze the images.

For further validation of primary spermatocytes by immunofluorescence analysis, isolated spermatogenic cells from different fractions were seeded on confocal dishes overnight, fixed in paraformaldehyde (4%) and blocked with the same blocking buffer used for cryosections. The spermatocyte specific marker γH2AX was incubated with isolated spermatogenic cells overnight at 4°C. Fluorescence-conjugated secondary antibodies were incubated with the samples for 1 h at room temperature. DAPI was used to stain the cell nuclei, and images were captured using a confocal fluorescence microscopy.

Statistical analyses

All the results in the present study were expressed as the means ± standard deviations (SDs). SPSS 17.0 (Chicago, USA) was used to perform an independent Student’s t-test for comparisons between two groups and one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls multiple comparison test for comparisons between three or more groups. Values with p < 0.05 were considered statistically significant.

Results

AA-induced reproductive damage in male pubertal mice

Mice were exposed to saline or AA (200 mg/L) in drinking water for four consecutive weeks. At the study endpoint, the testis and epididymis organ coefficients were significantly decreased by AA treatment (p < 0.01) (Figure 1(a)). The results of CASA are illustrated in Figure 1(b): in comparison with the control group, the AA-treated group showed significant reductions in total sperm count, motile cell percentage, and progressive cell percentage (p < 0.01 for all). These findings indicate that AA disturbs spermatogenesis. In addition, testicular and epididymal morphologies were evaluated to confirm this hypothesis. As shown in Figure 1(c), in the control group the morphology and structure of the testis and epididymis were normal in Leydig cells, Sertoli cells, and at various germ cell stages and spermatozoa were precisely organized. However, AA induced the dislocation of immature germ cells, vacuolization of the seminiferous epithelium, and reduction of cell layers in the testis. A significant decrease in epididymal sperm density was observed in mice treated with AA. The expression of the germ cell marker MVH was significantly downregulated by AA treatment (p < 0.05) (Figure 1(d)). In addition, although no significant difference was observed between the two groups, the expression of the proliferation marker PCNA showed a decreasing trend in AA-treated mice. Collectively, these data indicate that AA disrupts the seminiferous tubules and induces spermatogenic damage in pubertal mice.

Figure 1.

Effects of AA on relative organ weight, sperm parameters and morphological changes in the testis and epididymis of male pubertal mice. (a) Relative organ weight of the testis and epididymis. (b) Sperm count and sperm motility. (c) Representative H&E images of the testis and epididymis. Scale bar = 100 μm for typified field and scale bar = 50 μm for magnification of boxed area. The black arrow indicates the vacuolization of seminiferous epithelium. The black triangle indicates the reduction of cell layers in the testis. The blue triangle indicates the decreased density of epididymal sperm. (d) The expression levels of the germ cell marker MVH and proliferation marker PCNA in the testis. The data are expressed as the mean ± SD of ten mice in each group for annotations A and B, and three mice in each group for annotation D. *p < 0.05, **p < 0.01, compared with the control group.

AA-induced meiotic arrest in pubertal mice and spermatocytes

Testicular cells can be divided into three groups by flow cytometric analysis, according to their DNA content: (1) 1C cells are mainly spermatids; (2) 2C cells are pools of somatic cells, spermatogonia and secondary spermatocytes; and (3) 4C cells are typically primary spermatocytes. As shown in Figure 2(a) and (b), compared to mice in the control group, AA-treated mice showed a markedly increased proportion of 4C cells (p < 0.01) and a corresponding reduction in the percentage of 1C cells. There were no significant differences in the number of 2C cells between the groups. In addition, the localization and expression of SYCP3, a hallmark of the synaptonemal complex in spermatocytes, was significantly downregulated by AA in pubertal mice (p < 0.05) (Figure 2(c) and (d)). The expression of the meiotic regulating factors CyclinA1 and CDK2 was also decreased by AA treatment (p < 0.05) (Figure 2(d)). These results indicate that AA induced the arrest of meiotic prophase I in pubertal mice. To further validate the detrimental effects of AA on meiotic progression, primary spermatocytes were isolated and treated with AA. γH2AX staining showed that primary spermatocytes were isolated with high purity (average purity, 75.9%) and high cell viability (average, 89.3%). Representative images of the isolated spermatocytes are shown in Figure S1. No obvious cytotoxic effects were identified using 100 μM and 500 μM AA (Figure S1(c)). These concentrations were therefore selected and used in subsequent experiments. Similar to the results in pubertal mice, the expression of SYCP3, Cyclin A1 and CDK2 in isolated spermatocytes was downregulated by AA treatment (p < 0.01) (Figure 2(e)).

Figure 2.

Effects of AA on proportions of testicular cell populations and expression of meiotic proteins. (a) Representative fluorescent cell sorting images of flow cytometric analysis of testicular cells. (b) The percentage of 1C, 2C and 4C testicular cells by flow cytometric analysis. (c) Representative immunofluorescence images of SYCP3 staining in the testis. (d) and (e) The expression levels of meiotic regulating factors (SYCP3, CDK2 and Cyclin A1) in the testis and isolated spermatocytes, respectively. The data are expressed as the mean ± SD of three mice in each group for annotations B and D, and are expressed as the mean ± SE of three separate experiments with triplicate samples for annotation E. *p < 0.05, **p < 0.01, compared with the control group.

AA-induced excessive meiotic DSBs in pubertal mice and spermatocytes

Double-strand breaks are essential events during meiosis; however, persistent unrepaired meiotic DSBs or the accumulation thereof may lead to delayed meiosis. As shown in Figure 3(a), in control mice, intense γH2AX immunostaining was detected only in the sex body of spermatocytes at the pachytene stage. However, the spermatocytes obtained from AA-treated mice exhibited a significant increase in γH2AX foci in several autosomes. Immunoblot analysis showed that the expression of meiotic DSB signaling proteins (γH2AX, p-CHK2 and p-ATM) was significantly increased by AA treatment (p < 0.05 or p < 0.01) (Figure 3(b)). The results from the isolated spermatocytes also showed that AA treatment significantly elevated the levels of γH2AX, p-CHK2 and p-ATM (p < 0.05 or p < 0.01) (Figure 3(c)).

Figure 3.

Effects of AA on the meiotic process of pachytene spermatocytes and the expression of meiotic DSB signaling proteins. (a) Representative immunofluorescence images of SYCP3 and γH2AX staining in the pachytene spermatocytes of the testis. (b) and (c) The expression levels of meiotic DSB signaling proteins (γH2AX, p-ATM and p-CHK2) in the testis and isolated spermatocytes, respectively. The data are expressed as the mean ± SD of three mice in each group for annotation B and expressed as the mean ± SE of three separate experiments with triplicate samples for annotation C. *p < 0.05, **p < 0.01, compared with the control group.

Protective effect of resveratrol on AA-induced meiotic disruption in spermatocytes

The isolated spermatocytes were treated with resveratrol and AA in vitro to explore the effects of resveratrol on AA-induced meiotic disruption. As shown in Figure S2, resveratrol significantly attenuated AA-induced cytotoxicity in isolated spermatocytes (p < 0.05). In addition, AA-related inhibition of meiotic-regulating factors (SYCP3, CyclinaA1 and CDK2) and activation of meiotic DSB signaling proteins (γH2AX, p-CHK2 and p-ATM) were also rescued by resveratrol treatment (p < 0.01) (Figure 4). These results indicate the potential therapeutic role of resveratrol in AA-induced meiotic disruption.

Figure 4.

Protective effects of resveratrol on AA-induced meiotic disruption in spermatocytes. Expression levels of meiotic regulatory and DSB signaling proteins in isolated spermatocytes. The data are expressed as the mean ± SE of three separate experiments with triplicate samples. **p < 0.01, compared with the control group, ##p < 0.01, compared with the AA-treated group.

Discussion

In this study, we report persistent meiotic DSBs that mediate AA-induced impairment of spermatogenesis, especially meiotic deficits in pubertal mice. We further demonstrate that resveratrol offers a potential protective modality to relieve the deleterious effects of AA on testicular function. We focused on the adverse effects of AA on the male reproductive system in pubertal mice. These experiments were designed to simulate human exposure to AA during adolescence. Baked carbohydrate-rich food is the main source of AA exposure in the general population,³¹ and teenagers are a major subgroup that are likely to consume AA-contaminated snack products, such as salty sticks, cookies and morning cereals.¹⁰ It was estimated that children and adolescents consume approximately twice the amount of AA-rich food than that consumed by adults.³² In addition, the adolescent period is a critical developmental tage for the male reproductive system; during this stage, marked proliferation of germ cells, an increase in sex hormones and an increase in testis weight occur.³³ Studies investigating environmental toxicant-induced male reproductive toxicity during puberty have been performed extensively. For instance, pubertal exposure to a low dose of the endocrine disruptor zearalenone interfered with the meiosis process, reduced semen quality and compromised spermatogenesis through ER-mediated methylation pathways.³⁴ Fluoride decreased the organ coefficient of the epididymis, induced sperm abnormalities and decreased the expression of HSP-related genes in rats. Deleterious effects on the reproductive system were more evident in the pubertal period.³⁵ Therefore, in pubertal males the transitional development of the reproductive system is sensitive to xenobiotic-triggered testicular toxicity.

Laboratory evidence has demonstrated that AA exposure was correlated with impaired spermatogenesis, decreased epididymal sperm concentrations, poor semen quality and reduced fertility in male mice.^8,36 This was further validated by recent in vitro studies that showed that AA compromised cell viability, disrupted cell division and triggered genotoxicity in testis-associated Leydig, Sertoli and germ cells.^37–39 In this study, AA administration at 200 mg/L in drinking water significantly reduced the reproductive organ index (testis and epididymis), decreased sperm parameters (sperm count and motility), compromised the structure of the seminiferous epithelium and downregulated the expression of germ cell markers in pubertal mice, suggesting that AA disrupted spermatogenesis and that the laboratory model was established successfully. However, the mechanisms by which AA triggers testicular damage requires further investigation. Our previous study revealed the endocrine-disruptive effects of AA on sex hormones during puberty, which may contribute to its testicular toxicity.¹¹ In addition, recent studies have linked increased oxidative stress, inflammation, and apoptosis to AA-induced reproductive toxicity.^40,41 These studies were performed on whole testes and little is known regarding the potential testicular target cells of AA, such as spermatocytes, and their involvement in meiosis and spermatogenesis.

Spermatocytes and the complex process of meiosis play vital roles in the formation of haploid spermatids with two rounds of chromosome segregation.⁴² During meiosis, differentiating primary spermatocytes undergo meiotic synapsis, recombination, and segregation of homologous chromosomes. During this process gene transcription is extensively altered,⁴³ making meiosis a possible window of susceptibility for the toxicant-induced disruption of spermatogenesis.⁴⁴ In this study, we found that meiosis of primary spermatocytes was arrested in AA treated mice, as demonstrated by the significant increase in the proportion of 4C cells and decrease in 1C cells. This indicates that meiosis may be a vulnerable target for AA and play an essential role in AA-induced disruption of spermatogenesis. To test this hypothesis, the expression of meiotic markers and regulatory factors was further evaluated in vivo and in vitro. SYCP3 is a major component of the synaptonemal complex deposited on the chromosome axis, which attenuates the activity of RAD51 by promoting DMC1-mediated homologous recombination and plays a vital role in defining the chromosome architecture.^45,46 Spermatogenesis in sycp3^−/− male mice is disrupted by excessive apoptosis and failed meiotic progression in germ cells.⁴⁷ Cyclin A1 is a member of the mammalian A-type cyclin family, and forms an active kinase complex with its catalytic partner CDK2 in spermatocytes.⁴⁸ Studies have revealed the involvement of the CyclinA1-CDK2 complex, as a cell-cycle regulatory component, in regulating meiotic recombination. In addition, the disruption of CDK2 and CyclinA1 in transgenic mice was associated with infertility and reduced sperm production.^49,50 As illustrated in Figure 2, the expression levels of SYCP3, CDK2 and CyclinA1 were markedly decreased following AA exposure, suggesting that spermatocytes and meiosis may play a critical role in AA-induced testicular dysfunction.

Meiotic DSBs are major regulators of meiosis that stimulate homologous crossover recombination; nonetheless, increased, and persistent DSB signals on the autosomes of pachytene spermatocytes may also trigger checkpoint activation of kinases, resulting in the meiotic arrest of spermatocytes.⁵¹ The formation and repair of DSBs are modulated by three waves of H2AX phosphorylation (γH2AX) during meiosis. Physiologically, the γH2AX signal is only localized to the sex body at the pachytene stage of spermatocytes, whereas abnormal accumulation of γH2AX foci on the autosomes of pachytene spermatocytes is associated with arrested meiotic progression and chromosome abnormalities.⁵² Our results showed a marked increase in the γH2AX signal on the autosomes of pachytene spermatocytes following AA treatment, which supports the results from the flow cytometric analysis showing that meiosis was delayed by AA. The expression of the DNA damage response-related checkpoint kinases ATM/CHK2 is the main stress reaction of cells to DSBs in spermatocytes. This regulates the initiation of meiosis and the meiotic G2/M transition.⁵³ Concomitant with the persistent activation of γH2AX in the testes of pubertal mice and isolated spermatocytes, the phosphorylation of ATM and CHK2 was significantly upregulated following AA exposure, suggesting that a delay in the ATM/CHK2-mediated meiotic cycle may contribute to AA-induced arrest of meiosis.

Resveratrol is a polyphenol compound with antioxidant properties. Previous studies have revealed its beneficial effects against DNA damage triggered by reactive oxygen species, radiation, and mutagenic chemicals. For instance, resveratrol-mediated activation of the AMPK/SIRT7/HMGB1 pathway was involved in protection against radiation-induced cutaneous DNA damage.⁵⁴ Resveratrol can also activate SIRT3 and relieve asbestos-induced mitochondrial DNA damage in alveolar epithelial cells in mice.⁵⁵ In addition, resveratrol was reported to inhibit the activity of CYP2E1, which plays a critical role in the metabolism of AA to the more genotoxic compound GA. Therefore, considering that CYP2E1 is prominently expressed in spermatocytes during spermatogenesis and is essential for AA-induced reproductive toxicity,⁵⁶ it is plausible that the CYP2E1 inhibitor resveratrol could markedly rescue AA triggered persistent DSBs in spermatocytes and may serve as a potential therapeutic agent for AA-induced meiosis dysfunction. When using isolated primary spermatocytes, our results showed that resveratrol significantly abolished AA-related cytotoxicity, meiotic regulatory factor inhibition and meiotic DSB signaling protein activation.

Conclusion

In summary, the present study confirmed that pubertal AA exposure interferes with primary spermatocytes and delays meiosis, resulting in a ruptured testicular structure and decreased sperm count and motility. The increased γH2AX expansion patterns and ATM/CHK2-mediated persistent meiotic DSBs may be major contributors to the AA-induced arrest of meiosis. Furthermore, resveratrol treatment rescued meiotic regulation and DSB signaling factors and reversed the detrimental effects of AA exposure. This suggests that resveratrol treatment may be a reasonable approach to alleviate AA-related reproductive toxicity.

Supplemental Material

Supplemental Material - Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol

Supplemental Material for Acrylamide-induced meiotic arrest of spermatocytes in adolescent mice by triggering excessive DNA strand breaks: Potential therapeutic effects of resveratrol by Y Gao, D Zhang, P Wang, X Qu, J Xu, Y Yu and X Zhou in Human & Experimental Toxicology.

Footnotes

Author contributions

Yongyue Gao: Funding acquisition, Conceptualization, Methodology, Writing - original draft. Di Zhang: Methodology, Formal analysis, Project administration. Pu Wang: Methodology, Validation. Xiangling Qu: Methodology, Validation. Jiayi Xu: Project administration, Validation. Yongquan Yu: Funding acquisition, Writing - review & editing. Xunrong Zhou: Supervision, Project administration.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Key Disciplines of Traditional Chinese Medicine and Ethnic Medicine, the 14th Five-Year Plan of Guizhou [grant numbers QZYYZDXK (PY)-2021-07], and the National Natural Science Foundation of China [grant numbers 82003494].

ORCID iD

X Zhou

Supplemental Material

Supplemental material for this article is available online.

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