Protective Effect of Alpha Tocopherol Succinate on Al₂O₃-NPs Induced Damage in NMRI Mice Sertoli Cells: The Role of Inhibin B and Connexin 43

Protective Effect of Alpha Tocopherol Succinate on Al₂O₃-NPs Induced Damage in NMRI Mice Sertoli Cells: The Role of Inhibin B and Connexin 43 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Effect of Alpha Tocopherol Succinate on Al₂O₃-NPs Induced Damage in NMRI Mice Sertoli Cells: The Role of Inhibin B and Connexin 43 Milad Dadgar Naki, Elaheh Amini, Fatemeh Rohollah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7947162/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Molecular Biology Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background Sertoli cells are essential for spermatogenesis, controlling the microenvironment and interacting with germ cells. This study evaluates the preventive effect of alpha-tocopherol succinate against Al₂O₃-NPs-induced damage in NMRI mice Sertoli cells. Methods The effects of Al₂O₃-NPs and vitamin E were assessed on Sertoli cells using viability assays, oxidative stress markers, apoptosis detection, and gene expression analysis. Results Al₂O₃-NPs significantly reduced Sertoli cell viability and increased apoptosis, while vitamin E treatment mitigated these effects. Expression of INHBB and Cx43 was significantly downregulated by Al₂O₃-NPs and upregulated with vitamin E. Conclusion Vitamin E alleviates Al₂O₃-NPs-induced Sertoli cell damage by reducing oxidative stress, inhibiting apoptosis, and restoring INHBB and Cx43 expression. Alpha tocopherol succinate Alumina nanoparticles Sertoli cells Inhibin B Connexin 43 Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nanoparticles (NPs) have a wide range of applications in medicine due to their distinct characteristics over bulk materials [ 1 – 2 ]. Because of their small size, nanoparticles can move more easily from points of entry into the body to more delicate lymphatic and circulatory systems, where they can eventually find their way into bodily tissues and organs. There have been reports of accumulation in several organs, raising questions regarding systemic toxicity [ 3 ]. Aluminum (Al) is the most plentiful metal in the Earth’s crust with a high reactivity and tendency to combine with various compounds [ 4 ]. Based on data, exposure to aluminum (1-1000 micron) may cause oxidative stress, programmed cell death, and disturbance in the blood-brain barrier (BBB) [ 5 ]. Aluminum can trigger different disorders in male rats, including a thinner germinal epithelium with low spermatid and sperm counts in the lumen, motility, and viability [ 6 ]. Metal oxide nanoparticles (NPs), particularly aluminum oxide nanoparticles (Al₂O₃), are widely used in industry and domestic settings, prompting questions regarding their possible toxicity and environmental destiny [ 7 ]. Because of their unique physical and chemical properties, Al₂O₃-NPs have become more prevalent in a wide range of applications [ 8 ]. Regarding with toxicity of Al₂O₃-NPs, Mirshafa et al. (2018), demonstrated that oxidative stress may play a critical role in the toxicity of Al₂O₃-NPs after acute oral exposure [ 9 ]. Owing to the importance of reproductive system health, male reproductive toxicology aims to identify the potential adverse effects of chemical or biological agents on the function of cells and organs involved in male reproduction [ 10 ]. Previously, it was demonstrated the detrimental effects of Al₂O₃-NPs on the mice testis via MDA increment and attenuation of GSH and CAT levels [ 11 ]. Empirical data indicated that some nanoparticles may be able to pass through the BTB's theoretical barrier to prevent nanoparticle penetration and build-up inside the testis. Remarkably, it found that, mice testis absorbed small-sized silver nanoparticles (AgNPs) (22 and 42 nm) but not their larger counterparts (71 and 323 nm) after oral delivery. These findings are consistent with results from other animal and clinical studies emphasizing a negative correlation between increased concentrations of nanoparticles and reduced amount or quality of spermatozoa [ 12 ]. Recently, the identification of appropriate compounds possessing an effective potential for improving reproductive health was considered. In this regard, antioxidant compounds have pivotal roles such as inhibiting lipid peroxidation, and maintenance of germ cell structural integrity for proper male reproduction [ 13 ]. Vitamin E, the alpha-tocopherol succinate, is a dietary supplementation and the most effective fat-soluble antioxidant essential for reproduction, and the health of various organs. It is positioned as an efficient anti-toxic compound due to its capacity to neutralize free radicals and preserve cellular homeostasis [ 14 ]. The interaction of male germ cells and Sertoli cells via the paracrine and endocrine routes has been a pivotal function in male fertility [ 15 ]. Sertoli cells are a type of sustentacular cells that are associated pivotally during spermatogenesis and the development of the male reproductive system. [ 16 ]. In addition, Sertoli cells create the blood–testis barrier which divides the seminiferous tubules into a basal and adluminal compartments to separate the spermatogenesis process and avoid autoimmune disease and male infertility [ 17 ]. Numerous genes contribute to sexual organ development and function, and inhibin B has emerged prominently. Inhibin B is a male reproductive glycoprotein which is mainly secreted by Sertoli cells. This hormone is responsible for regulating FSH secretion from the pituitary gland through a negative feedback mechanism [ 18 ]. The levels of inhibin B as spermatogenesis serum marker is associated with sperm number, concentration and testicular size. In infertility, spermatogenic disruption is correlated with severe inhibin B attenuation along with FSH increment [ 19 ]. Connexin43 (Cx43) is the predominant member in family of transmembrane proteins which administered critical role in testicular development and function such as regulating testicular cell proliferation and differentiation, spermatogenic cell migration and death, Sertoli cells morphology and maturation [ 20 ]. Recent studies have proved the channel-independent roles of gap junctions, including their effects on multiple intracellular processes and the extrinsic guidance of migratory cells, which is mediated via Connexin (Cx)-mediated cell adhesion. Notably, Connexin 43 (Cx43) has become valuable for its effects on tubulin dynamics, cytoskeletal remodeling, and receptor signaling that affect cell migration [ 21 , 22 ]. Cx43 is the most common testicular Cx in all species except humans. Its protein localization in adult male testis with normal spermatogenesis and different types of spermatogenic impairment has been studied. Cx43 immunoreactivity is typically observed between Leydig cells (LCs), similar to what is seen in rodent testis. Additionally, within the seminiferous epithelium, Cx43 is essential for the formation of Sertoli cell (SC) junctional complexes, which form the anatomical basis of the blood-testis barrier (BTB) [ 23 , 24 ]. Related with the underlying mechanism of Al₂O₃-NPs toxicity in male reproduction especially in Sertoli cells there are rare information. Thus, the aim of this study was evaluation the possible mechanism of testicular damage induced by Al₂O₃-NPs and the preventive role of alpha-tocopherol succinate in toxicity associated with Al₂O₃-NPs in male supportive Sertoli cells in vitro and in vivo. Material and Method Isolation of NMRI mice Sertoli cells The animal intervention procedures in this study commenced after ethical acceptance secured from the Ethics Committee of Islamic Azad University of Tehran, Iran, with ethical code (IR.IAU.PS.REC.1402.557). To isolate Sertoli cells, 21-day-old NMRI mice were euthanized by cervical dislocation. The testicular tissue was surgically removed and carefully cleaned using PBS buffer (Gibco, Germany) and 5% Penicillin-Streptomycin (100X; Bio-Idea, Iran). The tissue was then divided into several pieces and incubated for 30 minutes with 1 mg/ml of collagenase type IV (Bio-Idea, Iran), % 0.25 Trypsin-EDTA (Bio-Idea, Iran), and gentamicin (Royan Company, Iran). The obtained homogenate was cultured in DMEM/F12 culture medium containing 10% FBS (Bio-IDEA, Iran), supplemented with L-glutamine (Sigma, Germany), followed by centrifugation, and transferred to a T75 flask. The cells were then incubated at 37°C with 5% CO2 in a Memmert incubator (Germany). The effect of Al₂O₃-NPs and alpha-tocopherol succinate on Sertoli cells viability In this study, aluminum oxide nanoparticle powder (Al₂O₃-NPs) (CAS Number 1344-28-1) and alpha-tocopherol succinate solution (Vitamin E) (Sigma-Aldrich, C₂₉H₅₀O₂) were used. The effects of these compounds on Sertoli cell viability were evaluated using the MTT assay. The study groups were as follows: 1. Control group: Sertoli cells in full culture media. 2. Experimental group 1: Sertoli cells exposed to varying concentrations of Al₂O₃-NPs (62.5, 125, 250, 500, and 1000 µg/ml) for 24, 48, and 72 hours. 3. Experimental group 2: Sertoli cells exposed to different concentrations of alpha-tocopherol succinate (6.25, 12.5, 25, 50, and 100 µg/ml) for 24, 48, and 72 hours. 4. Experimental group 3: Sertoli cells co-treated with Al₂O₃-NPs (500 µg/ml, IC₅₀ value) and alpha-tocopherol succinate at different concentrations for 48 hours. Following incubation, 50 µl of MTT (Merck, Germany) solution (0.5 mg/ml) was added to the wells and incubated for 4 hours at 37°C. Then, 50 µl of DMSO (Merck, Germany) was added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm using a Thermo ELISA reader , and cell viability was calculated using the formula: Cell viability (%) = \(\:\frac{Absorbance\:of\:treated\:cells}{Absorbance\:of\:control\:cells}\) ×100 Detection of apoptosis The effects of Al₂O₃-NPs and alpha-tocopherol succinate on Sertoli cells were evaluated using differential Acridine Orange/ Propidium Iodide (AO/PI) staining. In summary, the mice Sertoli cells exposed with the IC50 concentrations of alpha-tocopherol succinate (50 µg/ml) and Al₂O₃-NPs (500 µg/ml). After 48 hours of incubation, cells were harvested by trypsinization. Acridine orange (10 µl) was added to the cells, followed by 10 µl of propidium iodide and incubated for 5 min in the dark. A fluorescence microscopy (Olympus, Japan) was used to assess the alive, apoptosis and necrosis ratio. Detection of lipid peroxidation Sertoli cells were seeded in 6-well culture plates at a density of 2× 105 cells/well overnight. After incubation, the cells were pretreated with Al₂O₃-NPs and alpha-tocopherol succinate, alone or in combination, (50 and 500 µg/ml) for 48 hours. Then, the cells were collected, homogenized using a lysis buffer on ice, sonicated, centrifuged, and gently washed twice with PBS using the MDA assay kit (Abcam, United Kingdom). Eventually MDA concentration was detected in the supernatant using a microplate reader at a wavelength of 532 nm (Epoch, USA). INHBB and Cx43 mRNA detection by Real-time PCR A total of 106 Sertoli cells were cultured and administered 500 µg/ml Al₂O₃-NPs and 50 µg/l alpha-tocopherol succinate as well as a combination of doses, and after 48 hours of incubation total RNA was extracted using Karagen/Iran cell expansion solution. Using a Nanodrop, the amount of RNA was measured at a wavelength of 260 nm. Next, the SMOBIO single-stranded cDNA synthesis kit (England) was used to convert each sample's RNA into cDNA. Primer 3 Plus software was used to generate primers for the INHBB and Cx43 genes after nucleotide sequences were extracted from the NCBI database. Primer BLAST was used to confirm the secondary structure and specificity of primers. Table 1 displays primer sequences. The ACTB gene was selected as a reference gene, and expression analysis of the studied genes in different groups was conducted using SYBR Green Master Mix (Amplicon, Denmark) and the real-time device Step One Plus by QIAGEN. The thermal cycling profile used in this study was as follows: primary denaturation for 15 min at 95°C, denaturation for 15s at 95°C, annealing and extension for 1 min at 60°C, melt curve step1 for 15s at 95°C, melt curve step 2 for 1 min at 65°C, and melt curve step 3 for 15s at 95°C. Table 1 Sequence of primers Genes Primers Product size Tm (˚C) Cx43 Forward: 5’-ATGTCTGGGCACCTCTCTTTC-3’ Reverse: 5’-CCTAACTCTCCTTTTCCTTTGACTTC − 3’ 99bp 60 INHBB Forward: 5’-CCTGAAACTGCTCCCCTATGTC-3’ Reverse: 5’-GTCCACCTTCTTCTCCACCAC-3’ 118bp 60 ACTB Forward: 5'-GGCTGTATTCCCCTCCATCG-3' Reverse:5'- CCAGTTGGTAACAATGCCATGT-3' 154 bp 60 In vivo study and experimental design For this assay, 32 immature male NMRI mice (21-days-old) were obtained from Royan Institute, Iran. Mice were kept in standard conditions (temperature 25 ◦C, 12 h dark, and 12 h light) with adequate water and food. Mice under experiment were kept for one week according to the rules of the Ethics Committee of Islamic Azad University (IR.IAU.PS.REC.1402.557); and randomly divided into six groups (n = 8): Group 1: control mice which received intraperitoneal injection of 200 µl of ethanol 1%, Group 2: mice which received intraperitoneal injection of 100 mg/kg of alpha-tocopherol succinate, Group 3: mice which received intraperitoneal injection of 500 mg/kg of Al₂O₃-NPs, Group 4: mice which received intraperitoneal injection of 500 mg/kg of Al₂O₃-NPs along with 100 mg/kg of alpha-tocopherol succinate mixture. 14 days after injection, animals were sacrificed by cervical decapitation and their testicles were removed. The right testicles were used for histopathological assay while the left testicles were employed for real-time PCR examination. Histopathological assessment Testicles were fixed in Bouin’s solution for 24 hours. Then, they were carried out in a degree series of ethanol (70–100%), embedded in paraffin, and sectioned into 5 µm. After hematoxylin and eosin (H&E) staining, testicular changes were observed by an optical microscope (***). Quantitative real-time PCR (q-RT-PCR) RNA from testicular tissue was extracted and used to synthesize cDNA according to the manufacturer’s instructions. Next, the synthesized cDNA and related primers were evaluated by real-time PCR. The primer sequences are listed in Table 1 . The rate of changes in expression of target genes was compared to the control gene (ACTB). The "ΔΔCT" method was performed for this assay. Statistical analysis Three separate tests of interactions were used in this study to ensure accuracy and reproducibility. Data are presented as the mean ± standard deviation (SD). The data were first validated using Excel software, and significance was confirmed using GraphPad Prism 9. Tukey's multiple comparison test and analysis of variance were used in the statistical studies. P-value < 0.05 at the significance threshold was considered notable. Results Toxicity assessment As shown in Fig. 1 , Al₂O₃-NPs exerted dose dependent toxicity on Sertoli cells after 24, 48, and 72 hours (P < 0.05, P < 0.01, P < 0.001). In contrast, alpha-tocopherol succinate at concentrations of 50 µg/ml induced growth-promoting effects on Sertoli cells after 48 hours treatment, indicating their non-toxic nature. Notably, diverse concentrations of alpha-tocopherol succinate mitigated the toxicity induced by 500 µg/ml Al₂O₃-NPs in Sertoli cells (P < 0.05). Figure 1 The Sertoli cells morphological assessment Representative photomicrographs indicated morphological alterations, under exposure to Al₂O₃-NPs, alpha-tocopherol succinate and, Al₂O₃-NPs, along with alpha-tocopherol succinate. In the control, Sertoli cells possessed an epithelial-like shape. Under exposure to Al₂O₃-NPs in addition to attenuation in cell number, hallmarks of apoptosis have been observed. However, cell quantification in the simultaneous treatment group was greater than in the sample treated with Al₂O₃ nanoparticles alone, and in those cells with a less apoptotic appearance, after 48-hour treatment (Fig. 2 ). Figure 2 Evaluation of apoptosis, necrosis and alive cell percentage Figure 3 displays the images of alive, apoptosis and necrosis Sertoli cells after treatment with Al₂O₃-NPs and alpha-tocopherol succinate, alone or in combination. The alive cells (green color) were reduced and the proportion of cells under early apoptosis and secondary apoptosis (yellow and orange color) increased in Al₂O₃-NPs treated cells. In contrast, alpha-tocopherol succinate indicated no significant changes in alive cells proportion. Notably, cells exposed to Al₂O₃-NPs (IC50 value) and alpha-tocopherol succinate exhibited augmentation in the alive population, coupled with a reduction in apoptotic populations compared to the Al₂O₃-NPs alone. Figure 3 Stress oxidative assessment MDA concentration as a by-product of lipid peroxidation was used to evaluate oxidative stress after 48 h treatment. As shown in Fig. 4 , a markedly increment in the MDA level was observed at the Al₂O₃-NPs treated Sertoli cells compared with the control (P < 0.05), whereas, alpha-tocopherol succinate showed no increase in MDA concentration compared with the control (P < 0.001). On the other hand, combination treatment with Al₂O₃-NPs and alpha-tocopherol succinate in Sertoli cells significantly mitigated the MDA level as compared with Al₂O₃-NPs treatment alone (P < 0.001). Figure 4 Cx43 and INHBB gene expression in Sertoli cells The second aim of the present study was to determine the effects of Al₂O₃-NPs and alpha-tocopherol succinate on mice Sertoli cells mechanistically. As shown in Fig. 5 A, Cx43 gene expression evaluated in treated Sertoli cells compared to the control. Significantly, compared to the control, there was a 2.45-fold increase in Cx43 expression under alpha-tocopherol succinate treatment (P < 0.001). In contrast, there was a non-significant attenuation in Cx43 levels under the influence of Al₂O₃-NPs, with a 0.88-fold downregulation compared to the control (P:0.45). On the other hand, there was a substantial 1.74-fold increase in Cx43 gene expression in the treatment group exposed to Al₂O₃-NPs and alpha-tocopherol succinate concomitantly compared with the control (P < 0.05). In relation to INHBB mRNA expression, as revealed in Fig. 5 B, alpha-tocopherol succinate significantly up-regulated INHBB 2.75 times more to the control (P < 0.001). On the other hand, exposure to Al₂O₃-NPs significantly reduced the expression of INHBB 0.62 times compared with the control (P:0.002). Additionally, there was a 1.63-fold increase in INHBB mRNA levels after exposure to Al₂O₃-NPs and alpha-tocopherol succinate compared to the control (P < 0.05). Figure 5 Histological analysis of testes The results demonstrated the testes had a normal structure in the untreated mice, and all germ cell lineages were detectable normally. Treatment with alpha-tocopherol succinate had similar results with the control mice. However, treatment with LD50 concentration of Al₂O₃-NPs led to severe structural interruption of the testes, including weight and size reduction, disintegration in the germinal epithelium, and the appearance of empty spots inside the seminiferous tubules indicating damage to Sertoli cell junctions. In the co-treated group (treatment with Al₂O₃-NPs and alpha-tocopherol succinate), the mentioned interruptions were mild but different from the control. H&E results showed that Al₂O₃-NPs impaired the spermatogenesis in treated mice, and alpha-tocopherol succinate decreased these unfavorable effects on testes. Figure 6 Effect of Al₂O₃-NPs and alpha-tocopherol succinate on Cx43 and INHBB expression Figure 7 A shows the changes in Cx43 gene expression between treated and untreated Sertoli cells. Compared to the control, there is a remarkable up-regulation following treatment with alpha-tocopherol succinate (4.1-fold) (p ≤ 0.01). In contrast, there is a non-significant attenuation in gene expression after Al₂O₃-NPs treatment (0.68-fold). Also, there is a high increase in gene expression in Al₂O₃-NPs and alpha-tocopherol succinate treatment group (3.1-fold) (p ≤ 0.05). Figure 7 B shows the gene expression of INHBB between treated and control. Treatment with alpha-tocopherol succinate administered no significant changes in the INHBB gene expression. Al₂O₃-NPs greatly decreased INHBB expression (0.1-fold) (p ≤ 0.01). The gene expression in Al₂O₃-NPs and alpha-tocopherol succinate treated groups considerably declined (3.4-fold) (p ≤ 0.01). Figure 7 Discussion Recent studies have focused a great deal of attention on reproductive organ dysfunction brought on by various NPs [ 25 , 26 ]. Nevertheless, it is still unknown how to lessen the harmful effects of NPs on Sertoli as a testicular supportive cell. The current study is the first investigation of vitamin E's possible protection against Al₂O₃-NPs-induced toxicity in Sertoli cells. The findings of the MTT assay demonstrated that the toxicity of Al₂O₃-NPs to Sertoli cells rises in direct proportion to exposure time and NPs concentration. On the other hand, alpha-tocopherol succinate both reduced the toxicity of NPs and promoted Sertoli cell survival. Interestingly, there was a negative correlation between the toxicity of NPs and an increase in alpha-tocopherol succinate content after 48h treatment. The results of AO/PI staining showed that Al₂O₃-NPs increase primary and secondary apoptotic cells while decreasing the population of alive cells. On the other hand, alpha-tocopherol succinate had no discernible effect on the proportion of alive cells as compared to the control. In contrast, Sertoli cells exposed to alumina NPs and alpha-tocopherol succinate showed mitigation in the number of apoptotic cells and an elevation in the number of viable cells compared to Al₂O₃-NPs alone. According to Hamdi's research, testicular cells exposed to Al₂O₃-NPs experienced DNA damage, which led to severe histopathological changes and elevated levels of malondialdehyde (MDA) in testicular tissue. In the testicular tissue of rats given Al₂O₃-NPs, a significant fall in serum testosterone levels was also noted, coupled with a significant decrease in glutathione (GSH) and catalase (CAT) activity. On the other hand, co-administration of pumpkin seed oil improved histopathological changes, decreased MDA levels, strengthened antioxidant defenses in testicular tissue, and lessened DNA damage. Pumpkin seed oil (PSO) has gained popularity as a nutraceutical and as an edible oil. Prominent for its abundance in antioxidant vitamins, including α- and γ-tocopherol, β-carotene, and vitamin E [ 27 ], PSO plays a critical role in improving membrane fluidity and osmotic balance, thereby promoting intracellular and extracellular gaseous exchange because the existence of polyunsaturated fatty acid linoleic acid and oleic acid, a monounsaturated fatty acid, has been linked to a decrease in lipid peroxidation in the epididymis and testes [ 28 ]. According to Yu et al., vitamin E functions as a neuroprotectant against the neurotoxic effects of Al₂O₃-NPs against Caenorhabditis elegans neurotoxicity caused by Al₂O₃-NPs. However, vitamin E counteract neurotoxicity signs such as an aberrant axonal development, and death of neurons by protecting the nervous system's integrity and improving neuron health. Essentially, it suggests that vitamin E may be helpful in preventing the neurological consequences of other metal NPs [ 29 ]. It has been demonstrated that oral exposure to TiO2-NPs negatively affects viability, motility, and quantity of spermatozoa. Nonetheless, vitamin A and E supplementation showed promise in reducing these negative consequences, which is in line with our findings on vitamin E intake [ 30 ]. Mumtaz et al. demonstrated that vitamins C and E had therapeutic potential in preventing lead-induced reproductive organ damage [ 31 ]. Therefore, it seems that vitamin supplementation has the potential to shield spermatogenic lineage from harmful substances. In the current study, gene expression analysis showed that alpha-tocopherol succinate significantly up-regulated INHBB. On the other hand, Sertoli cells treated with alumina NPs showed a considerable mitigation of INHBB in transcriptional level. Interestingly, Sertoli cells treated with both Al₂O₃-NPs and alpha-tocopherol succinate showed INHBB up-regulation, suggesting that vitamin E administered an ameliorative effect against the toxicity of alumina NPs. Increased expression of INHBB generally indicates healthy testicles and normal functioning of Sertoli cells, which are associated with robust sperm production and eventually male fertility. On the other hand, decreased expression of INHBB can indicate impaired function of the testicles and deficiency of male reproduction. Based on literatures, the level of inhibin B have oscillation during development time in males. Inhibin B rises fivefold in normal rats to post-natal peak in termination of 2-weeks, then plateaued and decreased. Nevertheless, the levels of inhibin elevated due to reactivation of hypothalamic–pituitary–gonadal axis in puberty to reach (60% of 10–15 days old). In mice the content of inhibin B increased from post-natal day of 18 onward. Therefore, Sertoli cells in the prepubertal stage are currently used to assess the effect of various chemicals [ 32 – 34 ]. In normal condition, the high level of inhibin B is associated with normal fertility, while its attenuation is indicative of spermatogenic cell depletion. On the other hand, an abnormal elevation of inhibin B as well as a decrease of FSH release, exhibit germ cell injury [ 35 , 36 ]. Rossy et al. in 2020 reported that Bisphenol A as an endocrine disruptor increased the level of inhibin B in isolated mice Sertoli cells (7 day old) mainly due to an impact in hypothalamic-pituitary-gonadal axis [ 37 ]. Zhao et al., 2024 paved that Huangqi-Guizhi-Wuwutang (HGW) as a traditional Chinese product administered its protective effect via ameliorating cyclophosphamide-induced testicular injury, elevating hormonal level such as testosterone and INHBB and attenuating FSH and LH [ 38 ]. Related with Cx43, the gene expression findings indicated that Sertoli cells received alpha-tocopherol succinate showed a significant increase in the expression of the Cx43 when compared to the control group. In contrast, Cx43 expression showed no significant change in Al₂O₃-NPs -treated cells than in the control. Interestingly, Cx43 gene expression augmented in Sertoli cells received both Al₂O₃-NPs and alpha-tocopherol succinate than in the control. This suggests that alpha-tocopherol succinate has a protective effect against the toxicity induced by Al₂O₃-NPs. Connexin43 acts as a gap junctional protein between adjacent Sertoli cells along with Sertoli cells and spermatogenic cells responsible for coordination of spermatogenesis. Brehm et al. reported that connexin43 knockout in Sertoli cells is associated with testicular damage via spermatogenic arrest and Sertoli cell only syndrome. There are documents that Cx43 can alter under exposure with various chemical toxicants via disturbing functional mechanisms [ 39 , 40 ]. Kidder et al. elucidated that elimination of Cx43 in prenatal period led to an attenuation of spermatogenic cells, while in puberty, loss of Cx43 lead to spermatogenesis disruption, Leydig cell hyperplasia and declined sperm motility. It was demonstrated that the transcriptional and translational expression of Cx43 is noticeably disrupted in Sertoli cells of infertile men with secretory azoospermia. Li et al. paved that overexpression of Cx43 in Sertoli cells alleviated the disturbance of gap junction communication induced by Perfluorooctanesulfonate which acts as an environmental toxicant [ 41 – 43 ]. Regarding with the protective effect of natural compounds on testicular injury, in 2019, it was elucidated that flavonoid compounds such as luteolin administered the palliative role against triptolide testicular damage mainly via recruitment of Nrf2 translocation, elevation of antioxidant enzyme levels, apoptosis suppression, and increment the blood-testis barrier (BTB) integrity by Cx43 upregulation [ 44 ]. Zhao's research data supported our results indicating the protective effect of antioxidant compounds against chemical materials. It elucidated that lycopene (LYC) shields the blood-testis barrier (BTB) from the damaging effects of diethylhexyl phthalate (DEHP), allowing spermatogenesis to proceed normally. LYC was used in subsequent in vitro investigations to ameliorate the disruption of intercellular connections in mouse Sertoli cells (TM4 cells) and mouse Spermatogonia cells (GC-1 cells) caused by mono-(2-ethylhexyl) phthalate (MEHP). Both LYC therapy and connexin-43 (Cx43) overexpression enhanced cell migratory capacity and restored BTB integrity in MEHP-induced BTB impairment models of Sertoli cells [ 45 ]. Conclusion To our knowledge, the results of the current study disclosed that alpha-tocopherol succinate could alleviate damage induced by Al₂O₃-NPs on mice Sertoli cells via stress oxidative prevention, apoptosis suppression, and enhancement of inhibin B and Connexin43 expression. Preclinical analysis demonstrated that alpha-tocopherol succinate administered its protective effect via reduction of the testicular disturbance and elevation of spermatozoa number as well as up-regulation of inhibin B and Connexin43 at transcriptional level. Declarations Acknowledgments We would also like to thank GenIran Lab, Tehran, Iran, Central laboratory of Kharazmi University, and the Department of Molecular Cell Biology, for sharing their pearls of wisdom with us during the course of this thesis. Author contributions E.A. designed and directed the project, also analysed the spectra and statistics, and corrected the article. M.DN. performed the experiments and wrote the primary article draft; F.R. analysed the spectra and statistics and corrected the article. All authors have contributed to the final manuscript. Funding This work was not supported by research, writing, or publication. Data Availability Data available from the corresponding author upon reasonable request. Ethics approval statement Clinical trial number: not applicable. All animal procedures in this study commenced after ethical acceptance secured from the Ethics Committee of Islamic Azad University of Tehran, Iran, with ethical code (IR.IAU.PS.REC.1402.557). Conflict of interest All authors declare that they have no conflicts of interest and agree with the content of the manuscript. References Xuan L, Ju Z, Skonieczna M, Zhou PK, Huang R. 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Zhao Y, Wu J, Li X, Chen Q, Hong Z, Zheng L, et al. Protective effect of Huangqi-Guizhi-Wuwutang against cyclophosphamide-induced spermatogenesis dysfunction in mice by promoting steroid hormone biosynthesis. J Ethnopharmacol. 2024; 319:117260. doi: 10.1016/j.jep.2023.117260. Brehm R, Zeiler M, Rüttinger C, Herde K, Kibschull M, Winterhager E, et al. A Sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. Am J Pathol. 2007;171(1):19-31. doi:10.2353/ajpath.2007.061171. Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction. 2003;125(6):769-84. Walker WH. Androgen actions in the testis and the regulation of spermatogenesis. Adv Exp Med Biol. 2020; 1275:175-203. doi:10.1007/978-3-030-47492-7_8. Pointis G, Fiorini C, Defamie N, Gilleron J, Carette D, Valette A, et al. Connexins and spermatogenesis: lessons from knockout mice. J Androl. 2010;31(6):521-31. doi:10.2164/jandrol.110.010934. Chen H, Ge R-S, Zirkin BR. Leydig cells: from stem cells to aging. Mol Cell Endocrinol. 2009;306(1-2):9-16. doi: 10.1016/j.mce.2009.02.013. Oliveira PF, Sousa M, Silva BM, Monteiro MP, Alves MG. Obesity, energy balance and spermatogenesis. Reproduction. 2017;153(4): R173-85. doi:10.1530/REP-16-0526. Mínguez-Alarcón L, Chiu Y-H, Gaskins AJ, Minguez-Alarcon L, Williams PL, Ford JB, et al. Dietary fat intake in relation to reproductive hormones and testicular function in young men. Hum Reprod. 2017;32(12):2522-31. doi:10.1093/humrep/dex323. Rojas-García PP, Hernández-Carretero A, Gutierrez-Hernandez JM, Morales-Sánchez M, Álvarez-Rojas J, Zapata-Pérez A, et al. Dietary antioxidants and male fertility: a systematic review of randomized controlled trials. Nutrients. 2021;13(5):1789. doi:10.3390/nu13051789. Feng Y, Nasioudis D, Izvolsky KI, Wang Y, Fok KL, Lian Q, et al. The emerging role of Wnt signaling in blood-testis barrier dynamics and spermatogenesis. Semin Cell Dev Biol. 2020; 97:141-49. doi: 10.1016/j.semcdb.2019.06.008. Gervasi MG, Visconti PE. Molecular changes and signaling events occurring in spermatozoa during epididymal maturation. Andrology. 2017;5(2):204-18. doi:10.1111/andr.12366. Bonde JP, Flachs EM, Rimborg S, Glazer CH, Giwercman A, Ramlau-Hansen CH, et al. The epidemiologic evidence linking exposure to perfluoroalkyl substances (PFAS) with male reproductive health. Hum Reprod Update. 2022;28(1):151-76. doi:10.1093/humupd/dmab034. Barakat R, Lin PC, Park CJ, Best-Popescu C, Kannan K, Patisaul HB. Per- and polyfluoroalkyl substances (PFAS) disrupt estradiol-dependent signaling and uterine homeostasis in mice. Toxicol Sci. 2022;186(1):19-30. doi:10.1093/toxsci/kfab099. Sun B, Ma C, Liang J, Li Y, Li J, Sun C, et al. Single-cell RNA sequencing reveals spermatogenesis disorders induced by environmental endocrine disruptors. Cell Rep. 2022;38(6):110334. doi: 10.1016/j.celrep.2022.110334. Morrow CMK, Mruk DD, Cheng CY, Hess RA. Claudin and occluding expression and function in the seminiferous epithelium. Philos Trans R Soc Lond B Biol Sci. 2010;365(1546):1679-96. doi:10.1098/rstb.2009.0251. Xie J, Wang W, Wu S, Zhang H, Lin Y, Liu W, et al. The impact of paternal exposure to environmental pollutants on offspring development and health. Sci Total Environ. 2023; 891:164552. doi: 10.1016/j.scitotenv.2023.164552. Gore AC, Chappell VA, Fenton SE, Flaws JA, Nadal A, Prins GS, et al. Executive summary to EDC-2: The endocrine society's second scientific statement on endocrine-disrupting chemicals. Endocr Rev. 2015;36(6):593-602. doi:10.1210/er.2015-1093. Nassar GN, Al-Hiyari T, Yassin MA. The role of vitamin E in preventing oxidative stress-induced damage to sperm DNA integrity. Reprod Toxicol. 2021; 103:57-64. doi: 10.1016/j.reprotox.2021.04.005. Stevenson JC, Hodis HN, Pickar JH, Lobo RA. Coronary heart disease and the menopause transition: a review. Climacteric. 2021;24(6):556-64. doi:10.1080/13697137.2021.1949569. Rahman MS, Kwon WS, Pang MG. Prediction of male fertility using capacitation-associated proteins in spermatozoa. Mol Reprod Dev. 2017;84(9):749-59. doi:10.1002/mrd.22819. Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 1989;23(1):77-90. doi:10.1002/mrd.1120230108. Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod. 2011;26(7):1628-40. doi:10.1093/humrep/der132. Peltola V, Huhtaniemi I, Metsa-Ketela T, Ahotupa M. Induction of lipid peroxidation during steroidogenesis in the rat testis. Endocrinology. 1996;137(1):105-12. doi:10.1210/endo.137.1.8536639. Lysiak JJ, Turner SD, Nguyen QA, Singbartl K, Ley K, Turner TT. Essential role of leukocyte recruitment and cytokines in testicular ischemia-reperfusion injury. Biol Reprod. 2001;65(3):1418-25. doi:10.1095/biolreprod65.5.1418. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system for the morphometric and motility assessment of human spermatozoa. Mol Hum Reprod. 1996;2(4):299-310. doi:10.1093/molehr/2.4.299. Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction. 2003;125(6):769-84. Walker WH. Androgen actions in the testis and the regulation of spermatogenesis. Adv Exp Med Biol. 2020; 1275:175-203. doi:10.1007/978-3-030-47492-7_8. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.zip Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 09 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 25 Nov, 2025 Reviewers agreed at journal 29 Oct, 2025 Reviewers agreed at journal 29 Oct, 2025 Reviewers invited by journal 29 Oct, 2025 Editor assigned by journal 28 Oct, 2025 Submission checks completed at journal 28 Oct, 2025 First submitted to journal 25 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7947162","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":541934691,"identity":"99216e1b-954b-4940-9f63-b73a5a3f3ea6","order_by":0,"name":"Milad Dadgar Naki","email":"","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Milad","middleName":"Dadgar","lastName":"Naki","suffix":""},{"id":541934692,"identity":"5d4a806f-705a-46be-8e0e-2a8f6cb5b865","order_by":1,"name":"Elaheh Amini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3PIQvCQBTA8TcOtnK6ejLETyCcCGpQ/CobgmnZPBBuRbHqFxkGw8YD08BqMLiyZJhFNAhuimjxxGa4Pxw8Dn68OwCV6h8zQAfNywcCEGZh+Li1ZYS8kWj+Gylm+iSyTELS/XEJtbpfirAb7xzPwD0kq8+kMtbbjUUMjQDLNrrb1PHokIOdfiYcoWWVBGgBUo5uho4Hbv4XyQP7aJysq4D+nXQKYh7khBPasjQBzp3ANifsyxaGdFSZCjYoSDSJsSlYykMZMX0/YBfR7QWbuJmd11idmYMkOUvIc9lr1PPzHahUKpVK2g1Bt1cD+3O9KQAAAABJRU5ErkJggg==","orcid":"","institution":"Kharazmi University","correspondingAuthor":true,"prefix":"","firstName":"Elaheh","middleName":"","lastName":"Amini","suffix":""},{"id":541934693,"identity":"f6a92711-4232-4334-b0ab-ffa999590a47","order_by":2,"name":"Fatemeh Rohollah","email":"","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Rohollah","suffix":""}],"badges":[],"createdAt":"2025-10-27 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10:51:59","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116188,"visible":true,"origin":"","legend":"","description":"","filename":"99bcebc31e3f4991819f81ad5426d4191structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/0e8bd56771cda86bd8062d6b.xml"},{"id":95537226,"identity":"631e4c94-22b4-4a76-b938-a3d53aad5da5","added_by":"auto","created_at":"2025-11-10 10:51:59","extension":"html","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126506,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/6a535fa2446af15677962707.html"},{"id":95537215,"identity":"cb5eac17-0359-4b5a-8256-1c4a6502552b","added_by":"auto","created_at":"2025-11-10 10:51:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111485,"visible":true,"origin":"","legend":"\u003cp\u003eSertoli cell survival graph after treatment with Al₂O₃-NPs, alpha tocopherol succinate and combination treatment with Al₂O₃-NPs and different concentrations of alpha-tocopherol succinate. Data are reported as Mean ± SD. * P\u0026lt;0.05, ** P\u0026lt;0.01, and *** P\u0026lt;0.001 were considered significant.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/854ae9ed9781d54992ddcc17.jpg"},{"id":95537218,"identity":"5828b611-6408-446a-8e34-8b80de7172e6","added_by":"auto","created_at":"2025-11-10 10:51:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":157801,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of Al₂O₃-NPs, alpha tocopherol succinate and combination treatment with Al₂O₃-NPs and alpha-tocopherol succinate on Sertoli cell morphology. A: An untreated Sertoli cell (control); B. Sertoli cells treated with alpha-tocopherol succinate (50 μg/ml) indicated no obvious morphological changes after 48h treatment compared to the control. C. Sertoli cells treated with Al₂O₃-NPs (500 μg/ml) revealed an apoptotic appearance and cell confluence reduction after 48 hours of treatment. D. The synchronous treatment with a concentration of 500 μg/ml Al₂O₃-NPs and alpha-tocopherol succinate (concentration of 50 μg/ml), indicating reductive effect on damages induced by Al₂O₃-NPs on Sertoli cells.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/9cf3f8d18d8621d9ee1d98f5.jpg"},{"id":95654321,"identity":"f41a3b76-e942-4c71-aacd-a4fcd73a23da","added_by":"auto","created_at":"2025-11-11 16:11:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83305,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of AO/PI staining following Al₂O₃-NPs and alpha tocopherol succinate, treatment to Sertoli cells. A: The control, B: alpha-tocopherol succinate treatment alone, C: Al₂O₃-NPs treatment alone, and D: Al₂O₃-NPs along with alpha tocopherol succinate treatment.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/3e7cb12c869d5c844549780d.jpg"},{"id":95537217,"identity":"da609233-dacc-480b-a27e-0ddaf716660c","added_by":"auto","created_at":"2025-11-10 10:51:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23580,"visible":true,"origin":"","legend":"\u003cp\u003eThe levels of malondialdehyde (MDA) as oxidative stress index in the Sertoli cells after 48 h treatment with Al₂O₃-NPs and alpha-tocopherol succinate alone and in combination. Data are inserted as mean ± SD. * P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001 were considered statistically remarkable.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/909645a60efe2165a781abb2.jpg"},{"id":95654499,"identity":"0655f19a-2104-48d1-b7fc-f5132714e8ed","added_by":"auto","created_at":"2025-11-11 16:12:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55148,"visible":true,"origin":"","legend":"\u003cp\u003eRelative fold-change calculated by real-time PCR from critical Sertoli function involved genes in the control and cells treated with Al₂O₃-NPs and alpha-tocopherol succinate alone and in combination. A: Cx43 expression. B: INHBB expression in the study groups. The data obtained were normalized to the β-actin housekeeping gene and calculated relative to control. * P\u0026lt;0.05, and ** P\u0026lt;0.01 were denoted significant differences from the control.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/4cd98e6bda43f3ec27ff5781.jpg"},{"id":95537219,"identity":"c3716dd7-025b-43ba-a69e-eae619732f36","added_by":"auto","created_at":"2025-11-10 10:51:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":737696,"visible":true,"origin":"","legend":"\u003cp\u003eMicrographs of testicular tissue in treated and untreated groups after 2 weeks treatment with Al₂O₃-NPs and alpha-tocopherol succinate (alone and in combination). A. Control; B. Al₂O₃-NPs (500 mg/kg) treated mice; C. Vitamin E (alpha-tocopherol succinate; 100 mg/kg) treated mice; D. Al₂O₃-NPs (500 mg/kg) + Vitamin E (100 mg/kg), ×400. The asterisks exhibit normal spermatogenesis, and hollow black arrows indicate the disintegration of the testicular epithelium along with remarkable attenuation in the spermatogenic cells quantity.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/d248cc5d9c004b0c18be0acc.jpg"},{"id":95537222,"identity":"b90bbb32-536c-4683-90a6-7d7228f7235b","added_by":"auto","created_at":"2025-11-10 10:51:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":46965,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;qRT-PCR \u0026nbsp;analysis of the mRNA expression of Cx43 and INHBB in treated and untreated \u0026nbsp;Sertoli cells. In treatment groups, Sertoli cells treated with Al₂O₃-NPs and \u0026nbsp;alpha-tocopherol succinate alone and in combination for 48h. Data represented \u0026nbsp;as Mean ± SD. (p ≤ 0.05*) (p ≤0.01**).\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/1c54c13ae715dbdfafd89038.jpg"},{"id":104250654,"identity":"1537be36-0e39-4d1b-bf10-7344fd00e1f2","added_by":"auto","created_at":"2026-03-09 16:04:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2781141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/14b20295-11d8-4930-b5c7-9543a1fd0f3a.pdf"},{"id":95537229,"identity":"d8eab974-1ab8-4b6a-9ec7-17ceb76f9bb6","added_by":"auto","created_at":"2025-11-10 10:51:59","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15135253,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.zip","url":"https://assets-eu.researchsquare.com/files/rs-7947162/v1/dc74772d8c67cb556951317b.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Effect of Alpha Tocopherol Succinate on Al₂O₃-NPs Induced Damage in NMRI Mice Sertoli Cells: The Role of Inhibin B and Connexin 43","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanoparticles (NPs) have a wide range of applications in medicine due to their distinct characteristics over bulk materials [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Because of their small size, nanoparticles can move more easily from points of entry into the body to more delicate lymphatic and circulatory systems, where they can eventually find their way into bodily tissues and organs. There have been reports of accumulation in several organs, raising questions regarding systemic toxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAluminum (Al) is the most plentiful metal in the Earth\u0026rsquo;s crust with a high reactivity and tendency to combine with various compounds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Based on data, exposure to aluminum (1-1000 micron) may cause oxidative stress, programmed cell death, and disturbance in the blood-brain barrier (BBB) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Aluminum can trigger different disorders in male rats, including a thinner germinal epithelium with low spermatid and sperm counts in the lumen, motility, and viability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMetal oxide nanoparticles (NPs), particularly aluminum oxide nanoparticles (Al₂O₃), are widely used in industry and domestic settings, prompting questions regarding their possible toxicity and environmental destiny [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Because of their unique physical and chemical properties, Al₂O₃-NPs have become more prevalent in a wide range of applications [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Regarding with toxicity of Al₂O₃-NPs, Mirshafa et al. (2018), demonstrated that oxidative stress may play a critical role in the toxicity of Al₂O₃-NPs after acute oral exposure [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOwing to the importance of reproductive system health, male reproductive toxicology aims to identify the potential adverse effects of chemical or biological agents on the function of cells and organs involved in male reproduction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previously, it was demonstrated the detrimental effects of Al₂O₃-NPs on the mice testis via MDA increment and attenuation of GSH and CAT levels [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Empirical data indicated that some nanoparticles may be able to pass through the BTB's theoretical barrier to prevent nanoparticle penetration and build-up inside the testis. Remarkably, it found that, mice testis absorbed small-sized silver nanoparticles (AgNPs) (22 and 42 nm) but not their larger counterparts (71 and 323 nm) after oral delivery. These findings are consistent with results from other animal and clinical studies emphasizing a negative correlation between increased concentrations of nanoparticles and reduced amount or quality of spermatozoa [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecently, the identification of appropriate compounds possessing an effective potential for improving reproductive health was considered. In this regard, antioxidant compounds have pivotal roles such as inhibiting lipid peroxidation, and maintenance of germ cell structural integrity for proper male reproduction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Vitamin E, the alpha-tocopherol succinate, is a dietary supplementation and the most effective fat-soluble antioxidant essential for reproduction, and the health of various organs. It is positioned as an efficient anti-toxic compound due to its capacity to neutralize free radicals and preserve cellular homeostasis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe interaction of male germ cells and Sertoli cells via the paracrine and endocrine routes has been a pivotal function in male fertility [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Sertoli cells are a type of sustentacular cells that are associated pivotally during spermatogenesis and the development of the male reproductive system. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, Sertoli cells create the blood\u0026ndash;testis barrier which divides the seminiferous tubules into a basal and adluminal compartments to separate the spermatogenesis process and avoid autoimmune disease and male infertility [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNumerous genes contribute to sexual organ development and function, and inhibin B has emerged prominently. Inhibin B is a male reproductive glycoprotein which is mainly secreted by Sertoli cells. This hormone is responsible for regulating FSH secretion from the pituitary gland through a negative feedback mechanism [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The levels of inhibin B as spermatogenesis serum marker is associated with sperm number, concentration and testicular size. In infertility, spermatogenic disruption is correlated with severe inhibin B attenuation along with FSH increment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Connexin43 (Cx43) is the predominant member in family of transmembrane proteins which administered critical role in testicular development and function such as regulating testicular cell proliferation and differentiation, spermatogenic cell migration and death, Sertoli cells morphology and maturation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies have proved the channel-independent roles of gap junctions, including their effects on multiple intracellular processes and the extrinsic guidance of migratory cells, which is mediated via Connexin (Cx)-mediated cell adhesion. Notably, Connexin 43 (Cx43) has become valuable for its effects on tubulin dynamics, cytoskeletal remodeling, and receptor signaling that affect cell migration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Cx43 is the most common testicular Cx in all species except humans. Its protein localization in adult male testis with normal spermatogenesis and different types of spermatogenic impairment has been studied. Cx43 immunoreactivity is typically observed between Leydig cells (LCs), similar to what is seen in rodent testis. Additionally, within the seminiferous epithelium, Cx43 is essential for the formation of Sertoli cell (SC) junctional complexes, which form the anatomical basis of the blood-testis barrier (BTB) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRelated with the underlying mechanism of Al₂O₃-NPs toxicity in male reproduction especially in Sertoli cells there are rare information. Thus, the aim of this study was evaluation the possible mechanism of testicular damage induced by Al₂O₃-NPs and the preventive role of alpha-tocopherol succinate in toxicity associated with Al₂O₃-NPs in male supportive Sertoli cells in vitro and in vivo.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eIsolation of NMRI mice Sertoli cells\u003c/h2\u003e\n \u003cp\u003eThe animal intervention procedures in this study commenced after ethical acceptance secured from the Ethics Committee of Islamic Azad University of Tehran, Iran, with ethical code (IR.IAU.PS.REC.1402.557). To isolate Sertoli cells, 21-day-old NMRI mice were euthanized by cervical dislocation. The testicular tissue was surgically removed and carefully cleaned using PBS buffer (Gibco, Germany) and 5% Penicillin-Streptomycin (100X; Bio-Idea, Iran). The tissue was then divided into several pieces and incubated for 30 minutes with 1 mg/ml of collagenase type IV (Bio-Idea, Iran), % 0.25 Trypsin-EDTA (Bio-Idea, Iran), and gentamicin (Royan Company, Iran). The obtained homogenate was cultured in DMEM/F12 culture medium containing 10% FBS (Bio-IDEA, Iran), supplemented with L-glutamine (Sigma, Germany), followed by centrifugation, and transferred to a T75 flask. The cells were then incubated at 37\u0026deg;C with 5% CO2 in a Memmert incubator (Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eThe effect of Al₂O₃-NPs and alpha-tocopherol succinate on Sertoli cells viability\u003c/h3\u003e\n\u003cp\u003eIn this study, \u003cstrong\u003ealuminum oxide nanoparticle powder (Al₂O₃-NPs)\u003c/strong\u003e (CAS Number 1344-28-1) and \u003cstrong\u003ealpha-tocopherol succinate solution (Vitamin E)\u003c/strong\u003e (Sigma-Aldrich, C₂₉H₅₀O₂) were used.\u003c/p\u003e\n\u003cp\u003eThe effects of these compounds on Sertoli cell viability were evaluated using the MTT assay. The study groups were as follows:\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e1. Control group: Sertoli cells in full culture media.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e2. Experimental group 1: Sertoli cells exposed to varying concentrations of Al₂O₃-NPs (62.5, 125, 250, 500, and 1000 \u0026micro;g/ml) for 24, 48, and 72 hours.\u003c/p\u003e\n\u003c/span\u003e\u003cspan\u003e\n \u003cp\u003e3. Experimental group 2: Sertoli cells exposed to different concentrations of alpha-tocopherol succinate (6.25, 12.5, 25, 50, and 100 \u0026micro;g/ml) for 24, 48, and 72 hours.\u003c/p\u003e\n\u003c/span\u003e\u003cspan\u003e\n \u003cp\u003e4. Experimental group 3: Sertoli cells co-treated with Al₂O₃-NPs (500 \u0026micro;g/ml, IC₅₀ value) and alpha-tocopherol succinate at different concentrations for 48 hours.\u003c/p\u003e\n\u003c/span\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eFollowing incubation, 50 \u0026micro;l of MTT (Merck, Germany) solution (0.5 mg/ml) was added to the wells and incubated for 4 hours at 37\u0026deg;C. Then, 50 \u0026micro;l of DMSO (Merck, Germany) was added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm using a \u003cstrong\u003eThermo ELISA reader\u003c/strong\u003e, and cell viability was calculated using the formula:\u003c/p\u003e\n\u003cp\u003eCell viability (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Absorbance\\:of\\:treated\\:cells}{Absorbance\\:of\\:control\\:cells}\\)\u003c/span\u003e\u003c/span\u003e \u0026times;100\u003c/p\u003e\n\u003ch3\u003eDetection of apoptosis\u003c/h3\u003e\n\u003cp\u003eThe effects of Al₂O₃-NPs and alpha-tocopherol succinate on Sertoli cells were evaluated using differential Acridine Orange/ Propidium Iodide (AO/PI) staining. In summary, the mice Sertoli cells exposed with the IC50 concentrations of alpha-tocopherol succinate (50 \u0026micro;g/ml) and Al₂O₃-NPs (500 \u0026micro;g/ml). After 48 hours of incubation, cells were harvested by trypsinization. Acridine orange (10 \u0026micro;l) was added to the cells, followed by 10 \u0026micro;l of propidium iodide and incubated for 5 min in the dark. A fluorescence microscopy (Olympus, Japan) was used to assess the alive, apoptosis and necrosis ratio.\u003c/p\u003e\n\u003ch3\u003eDetection of lipid peroxidation\u003c/h3\u003e\n\u003cp\u003eSertoli cells were seeded in 6-well culture plates at a density of 2\u0026times; 105 cells/well overnight. After incubation, the cells were pretreated with Al₂O₃-NPs and alpha-tocopherol succinate, alone or in combination, (50 and 500 \u0026micro;g/ml) for 48 hours. Then, the cells were collected, homogenized using a lysis buffer on ice, sonicated, centrifuged, and gently washed twice with PBS using the MDA assay kit (Abcam, United Kingdom). Eventually MDA concentration was detected in the supernatant using a microplate reader at a wavelength of 532 nm (Epoch, USA).\u003c/p\u003e\n\u003ch3\u003eINHBB and Cx43 mRNA detection by Real-time PCR\u003c/h3\u003e\n\u003cp\u003eA total of 106 Sertoli cells were cultured and administered 500 \u0026micro;g/ml Al₂O₃-NPs and 50 \u0026micro;g/l alpha-tocopherol succinate as well as a combination of doses, and after 48 hours of incubation total RNA was extracted using Karagen/Iran cell expansion solution. Using a Nanodrop, the amount of RNA was measured at a wavelength of 260 nm. Next, the SMOBIO single-stranded cDNA synthesis kit (England) was used to convert each sample\u0026apos;s RNA into cDNA. Primer 3 Plus software was used to generate primers for the INHBB and Cx43 genes after nucleotide sequences were extracted from the NCBI database. Primer BLAST was used to confirm the secondary structure and specificity of primers. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e displays primer sequences. The ACTB gene was selected as a reference gene, and expression analysis of the studied genes in different groups was conducted using SYBR Green Master Mix (Amplicon, Denmark) and the real-time device Step One Plus by QIAGEN. The thermal cycling profile used in this study was as follows: primary denaturation for 15 min at 95\u0026deg;C, denaturation for 15s at 95\u0026deg;C, annealing and extension for 1 min at 60\u0026deg;C, melt curve step1 for 15s at 95\u0026deg;C, melt curve step 2 for 1 min at 65\u0026deg;C, and melt curve step 3 for 15s at 95\u0026deg;C.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSequence of primers\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProduct size\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTm (˚C)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCx43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForward: 5\u0026rsquo;-ATGTCTGGGCACCTCTCTTTC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse: 5\u0026rsquo;-CCTAACTCTCCTTTTCCTTTGACTTC \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINHBB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForward: 5\u0026rsquo;-CCTGAAACTGCTCCCCTATGTC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eReverse: 5\u0026rsquo;-GTCCACCTTCTTCTCCACCAC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACTB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForward: 5\u0026apos;-GGCTGTATTCCCCTCCATCG-3\u0026apos;\u003c/p\u003e\n \u003cp\u003eReverse:5\u0026apos;- CCAGTTGGTAACAATGCCATGT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e154 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vivo study and experimental design\u003c/h2\u003e\n \u003cp\u003eFor this assay, 32 immature male NMRI mice (21-days-old) were obtained from Royan Institute, Iran. Mice were kept in standard conditions (temperature 25 ◦C, 12 h dark, and 12 h light) with adequate water and food. Mice under experiment were kept for one week according to the rules of the Ethics Committee of Islamic Azad University (IR.IAU.PS.REC.1402.557); and randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;8): Group 1: control mice which received intraperitoneal injection of 200 \u0026micro;l of ethanol 1%, Group 2: mice which received intraperitoneal injection of 100 mg/kg of alpha-tocopherol succinate, Group 3: mice which received intraperitoneal injection of 500 mg/kg of Al₂O₃-NPs, Group 4: mice which received intraperitoneal injection of 500 mg/kg of Al₂O₃-NPs along with 100 mg/kg of alpha-tocopherol succinate mixture. 14 days after injection, animals were sacrificed by cervical decapitation and their testicles were removed. The right testicles were used for histopathological assay while the left testicles were employed for real-time PCR examination.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eHistopathological assessment\u003c/h3\u003e\n\u003cp\u003eTesticles were fixed in Bouin\u0026rsquo;s solution for 24 hours. Then, they were carried out in a degree series of ethanol (70\u0026ndash;100%), embedded in paraffin, and sectioned into 5 \u0026micro;m. After hematoxylin and eosin (H\u0026amp;E) staining, testicular changes were observed by an optical microscope (***).\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time PCR (q-RT-PCR)\u003c/h3\u003e\n\u003cp\u003eRNA from testicular tissue was extracted and used to synthesize cDNA according to the manufacturer\u0026rsquo;s instructions. Next, the synthesized cDNA and related primers were evaluated by real-time PCR. The primer sequences are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The rate of changes in expression of target genes was compared to the control gene (ACTB). The \u0026quot;\u0026Delta;\u0026Delta;CT\u0026quot; method was performed for this assay.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThree separate tests of interactions were used in this study to ensure accuracy and reproducibility. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The data were first validated using Excel software, and significance was confirmed using GraphPad Prism 9. Tukey\u0026apos;s multiple comparison test and analysis of variance were used in the statistical studies. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 at the significance threshold was considered notable.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eToxicity assessment\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Al₂O₃-NPs exerted dose dependent toxicity on Sertoli cells after 24, 48, and 72 hours (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, alpha-tocopherol succinate at concentrations of 50 \u0026micro;g/ml induced growth-promoting effects on Sertoli cells after 48 hours treatment, indicating their non-toxic nature. Notably, diverse concentrations of alpha-tocopherol succinate mitigated the toxicity induced by 500 \u0026micro;g/ml Al₂O₃-NPs in Sertoli cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eThe Sertoli cells morphological assessment\u003c/h2\u003e\n \u003cp\u003eRepresentative photomicrographs indicated morphological alterations, under exposure to Al₂O₃-NPs, alpha-tocopherol succinate and, Al₂O₃-NPs, along with alpha-tocopherol succinate. In the control, Sertoli cells possessed an epithelial-like shape. Under exposure to Al₂O₃-NPs in addition to attenuation in cell number, hallmarks of apoptosis have been observed. However, cell quantification in the simultaneous treatment group was greater than in the sample treated with Al₂O₃ nanoparticles alone, and in those cells with a less apoptotic appearance, after 48-hour treatment (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eEvaluation of apoptosis, necrosis and alive cell percentage\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e displays the images of alive, apoptosis and necrosis Sertoli cells after treatment with Al₂O₃-NPs and alpha-tocopherol succinate, alone or in combination. The alive cells (green color) were reduced and the proportion of cells under early apoptosis and secondary apoptosis (yellow and orange color) increased in Al₂O₃-NPs treated cells. In contrast, alpha-tocopherol succinate indicated no significant changes in alive cells proportion. Notably, cells exposed to Al₂O₃-NPs (IC50 value) and alpha-tocopherol succinate exhibited augmentation in the alive population, coupled with a reduction in apoptotic populations compared to the Al₂O₃-NPs alone.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eStress oxidative assessment\u003c/h2\u003e\n \u003cp\u003eMDA concentration as a by-product of lipid peroxidation was used to evaluate oxidative stress after 48 h treatment. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, a markedly increment in the MDA level was observed at the Al₂O₃-NPs treated Sertoli cells compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas, alpha-tocopherol succinate showed no increase in MDA concentration compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, combination treatment with Al₂O₃-NPs and alpha-tocopherol succinate in Sertoli cells significantly mitigated the MDA level as compared with Al₂O₃-NPs treatment alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eCx43 and INHBB gene expression in Sertoli cells\u003c/h2\u003e\n \u003cp\u003eThe second aim of the present study was to determine the effects of Al₂O₃-NPs and alpha-tocopherol succinate on mice Sertoli cells mechanistically. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, Cx43 gene expression evaluated in treated Sertoli cells compared to the control. Significantly, compared to the control, there was a 2.45-fold increase in Cx43 expression under alpha-tocopherol succinate treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, there was a non-significant attenuation in Cx43 levels under the influence of Al₂O₃-NPs, with a 0.88-fold downregulation compared to the control (P:0.45). On the other hand, there was a substantial 1.74-fold increase in Cx43 gene expression in the treatment group exposed to Al₂O₃-NPs and alpha-tocopherol succinate concomitantly compared with the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eIn relation to INHBB mRNA expression, as revealed in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, alpha-tocopherol succinate significantly up-regulated INHBB 2.75 times more to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, exposure to Al₂O₃-NPs significantly reduced the expression of INHBB 0.62 times compared with the control (P:0.002). Additionally, there was a 1.63-fold increase in INHBB mRNA levels after exposure to Al₂O₃-NPs and alpha-tocopherol succinate compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eHistological analysis of testes\u003c/h2\u003e\n \u003cp\u003eThe results demonstrated the testes had a normal structure in the untreated mice, and all germ cell lineages were detectable normally. Treatment with alpha-tocopherol succinate had similar results with the control mice. However, treatment with LD50 concentration of Al₂O₃-NPs led to severe structural interruption of the testes, including weight and size reduction, disintegration in the germinal epithelium, and the appearance of empty spots inside the seminiferous tubules indicating damage to Sertoli cell junctions. In the co-treated group (treatment with Al₂O₃-NPs and alpha-tocopherol succinate), the mentioned interruptions were mild but different from the control. H\u0026amp;E results showed that Al₂O₃-NPs impaired the spermatogenesis in treated mice, and alpha-tocopherol succinate decreased these unfavorable effects on testes.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of Al₂O₃-NPs and alpha-tocopherol succinate on Cx43 and INHBB expression\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the changes in Cx43 gene expression between treated and untreated Sertoli cells. Compared to the control, there is a remarkable up-regulation following treatment with alpha-tocopherol succinate (4.1-fold) (p\u0026thinsp;\u0026le;\u0026thinsp;0.01). In contrast, there is a non-significant attenuation in gene expression after Al₂O₃-NPs treatment (0.68-fold). Also, there is a high increase in gene expression in Al₂O₃-NPs and alpha-tocopherol succinate treatment group (3.1-fold) (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB shows the gene expression of INHBB between treated and control. Treatment with alpha-tocopherol succinate administered no significant changes in the INHBB gene expression. Al₂O₃-NPs greatly decreased INHBB expression (0.1-fold) (p\u0026thinsp;\u0026le;\u0026thinsp;0.01). The gene expression in Al₂O₃-NPs and alpha-tocopherol succinate treated groups considerably declined (3.4-fold) (p\u0026thinsp;\u0026le;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecent studies have focused a great deal of attention on reproductive organ dysfunction brought on by various NPs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Nevertheless, it is still unknown how to lessen the harmful effects of NPs on Sertoli as a testicular supportive cell. The current study is the first investigation of vitamin E's possible protection against Al₂O₃-NPs-induced toxicity in Sertoli cells.\u003c/p\u003e\u003cp\u003eThe findings of the MTT assay demonstrated that the toxicity of Al₂O₃-NPs to Sertoli cells rises in direct proportion to exposure time and NPs concentration. On the other hand, alpha-tocopherol succinate both reduced the toxicity of NPs and promoted Sertoli cell survival. Interestingly, there was a negative correlation between the toxicity of NPs and an increase in alpha-tocopherol succinate content after 48h treatment. The results of AO/PI staining showed that Al₂O₃-NPs increase primary and secondary apoptotic cells while decreasing the population of alive cells. On the other hand, alpha-tocopherol succinate had no discernible effect on the proportion of alive cells as compared to the control. In contrast, Sertoli cells exposed to alumina NPs and alpha-tocopherol succinate showed mitigation in the number of apoptotic cells and an elevation in the number of viable cells compared to Al₂O₃-NPs alone.\u003c/p\u003e\u003cp\u003eAccording to Hamdi's research, testicular cells exposed to Al₂O₃-NPs experienced DNA damage, which led to severe histopathological changes and elevated levels of malondialdehyde (MDA) in testicular tissue. In the testicular tissue of rats given Al₂O₃-NPs, a significant fall in serum testosterone levels was also noted, coupled with a significant decrease in glutathione (GSH) and catalase (CAT) activity. On the other hand, co-administration of pumpkin seed oil improved histopathological changes, decreased MDA levels, strengthened antioxidant defenses in testicular tissue, and lessened DNA damage. Pumpkin seed oil (PSO) has gained popularity as a nutraceutical and as an edible oil. Prominent for its abundance in antioxidant vitamins, including α- and γ-tocopherol, β-carotene, and vitamin E [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], PSO plays a critical role in improving membrane fluidity and osmotic balance, thereby promoting intracellular and extracellular gaseous exchange because the existence of polyunsaturated fatty acid linoleic acid and oleic acid, a monounsaturated fatty acid, has been linked to a decrease in lipid peroxidation in the epididymis and testes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccording to Yu et al., vitamin E functions as a neuroprotectant against the neurotoxic effects of Al₂O₃-NPs against Caenorhabditis elegans neurotoxicity caused by Al₂O₃-NPs. However, vitamin E counteract neurotoxicity signs such as an aberrant axonal development, and death of neurons by protecting the nervous system's integrity and improving neuron health. Essentially, it suggests that vitamin E may be helpful in preventing the neurological consequences of other metal NPs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt has been demonstrated that oral exposure to TiO2-NPs negatively affects viability, motility, and quantity of spermatozoa. Nonetheless, vitamin A and E supplementation showed promise in reducing these negative consequences, which is in line with our findings on vitamin E intake [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Mumtaz et al. demonstrated that vitamins C and E had therapeutic potential in preventing lead-induced reproductive organ damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, it seems that vitamin supplementation has the potential to shield spermatogenic lineage from harmful substances.\u003c/p\u003e\u003cp\u003eIn the current study, gene expression analysis showed that alpha-tocopherol succinate significantly up-regulated INHBB. On the other hand, Sertoli cells treated with alumina NPs showed a considerable mitigation of INHBB in transcriptional level. Interestingly, Sertoli cells treated with both Al₂O₃-NPs and alpha-tocopherol succinate showed INHBB up-regulation, suggesting that vitamin E administered an ameliorative effect against the toxicity of alumina NPs. Increased expression of INHBB generally indicates healthy testicles and normal functioning of Sertoli cells, which are associated with robust sperm production and eventually male fertility. On the other hand, decreased expression of INHBB can indicate impaired function of the testicles and deficiency of male reproduction.\u003c/p\u003e\u003cp\u003eBased on literatures, the level of inhibin B have oscillation during development time in males. Inhibin B rises fivefold in normal rats to post-natal peak in termination of 2-weeks, then plateaued and decreased. Nevertheless, the levels of inhibin elevated due to reactivation of hypothalamic\u0026ndash;pituitary\u0026ndash;gonadal axis in puberty to reach (60% of 10\u0026ndash;15 days old). In mice the content of inhibin B increased from post-natal day of 18 onward. Therefore, Sertoli cells in the prepubertal stage are currently used to assess the effect of various chemicals [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In normal condition, the high level of inhibin B is associated with normal fertility, while its attenuation is indicative of spermatogenic cell depletion. On the other hand, an abnormal elevation of inhibin B as well as a decrease of FSH release, exhibit germ cell injury [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRossy et al. in 2020 reported that Bisphenol A as an endocrine disruptor increased the level of inhibin B in isolated mice Sertoli cells (7 day old) mainly due to an impact in hypothalamic-pituitary-gonadal axis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Zhao et al., 2024 paved that Huangqi-Guizhi-Wuwutang (HGW) as a traditional Chinese product administered its protective effect via ameliorating cyclophosphamide-induced testicular injury, elevating hormonal level such as testosterone and INHBB and attenuating FSH and LH [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRelated with Cx43, the gene expression findings indicated that Sertoli cells received alpha-tocopherol succinate showed a significant increase in the expression of the Cx43 when compared to the control group. In contrast, Cx43 expression showed no significant change in Al₂O₃-NPs -treated cells than in the control. Interestingly, Cx43 gene expression augmented in Sertoli cells received both Al₂O₃-NPs and alpha-tocopherol succinate than in the control. This suggests that alpha-tocopherol succinate has a protective effect against the toxicity induced by Al₂O₃-NPs.\u003c/p\u003e\u003cp\u003eConnexin43 acts as a gap junctional protein between adjacent Sertoli cells along with Sertoli cells and spermatogenic cells responsible for coordination of spermatogenesis. Brehm et al. reported that connexin43 knockout in Sertoli cells is associated with testicular damage via spermatogenic arrest and Sertoli cell only syndrome. There are documents that Cx43 can alter under exposure with various chemical toxicants via disturbing functional mechanisms [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eKidder et al. elucidated that elimination of Cx43 in prenatal period led to an attenuation of spermatogenic cells, while in puberty, loss of Cx43 lead to spermatogenesis disruption, Leydig cell hyperplasia and declined sperm motility. It was demonstrated that the transcriptional and translational expression of Cx43 is noticeably disrupted in Sertoli cells of infertile men with secretory azoospermia. Li et al. paved that overexpression of Cx43 in Sertoli cells alleviated the disturbance of gap junction communication induced by Perfluorooctanesulfonate which acts as an environmental toxicant [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegarding with the protective effect of natural compounds on testicular injury, in 2019, it was elucidated that flavonoid compounds such as luteolin administered the palliative role against triptolide testicular damage mainly via recruitment of Nrf2 translocation, elevation of antioxidant enzyme levels, apoptosis suppression, and increment the blood-testis barrier (BTB) integrity by Cx43 upregulation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eZhao's research data supported our results indicating the protective effect of antioxidant compounds against chemical materials. It elucidated that lycopene (LYC) shields the blood-testis barrier (BTB) from the damaging effects of diethylhexyl phthalate (DEHP), allowing spermatogenesis to proceed normally. LYC was used in subsequent in vitro investigations to ameliorate the disruption of intercellular connections in mouse Sertoli cells (TM4 cells) and mouse Spermatogonia cells (GC-1 cells) caused by mono-(2-ethylhexyl) phthalate (MEHP). Both LYC therapy and connexin-43 (Cx43) overexpression enhanced cell migratory capacity and restored BTB integrity in MEHP-induced BTB impairment models of Sertoli cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo our knowledge, the results of the current study disclosed that alpha-tocopherol succinate could alleviate damage induced by Al₂O₃-NPs on mice Sertoli cells via stress oxidative prevention, apoptosis suppression, and enhancement of inhibin B and Connexin43 expression. Preclinical analysis demonstrated that alpha-tocopherol succinate administered its protective effect via reduction of the testicular disturbance and elevation of spermatozoa number as well as up-regulation of inhibin B and Connexin43 at transcriptional level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would also like to thank GenIran Lab, Tehran, Iran, Central laboratory of Kharazmi University, and the Department of Molecular Cell Biology, for sharing their pearls of wisdom with us during the course of this thesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.A. designed and directed the project, also analysed the spectra and statistics, and corrected the article. M.DN. performed the experiments and wrote the primary article draft; F.R. analysed the spectra and statistics and corrected the article. All authors have contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was not supported by research, writing, or publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable. All animal procedures in this study commenced after ethical acceptance secured from the Ethics Committee of Islamic Azad University of Tehran, Iran, with ethical code (IR.IAU.PS.REC.1402.557).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflicts of interest and agree with the content of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eXuan L, Ju Z, Skonieczna M, Zhou PK, Huang R.\u003c/strong\u003e Nanoparticles-induced potential toxicity on human health: Applications, toxicity mechanisms, and evaluation models. \u003cem\u003eMedComm.\u003c/em\u003e 2023;4(4): e327. doi:10.1002/mco2.327.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eWang H, Liu Z, Larsen M, Hastings R, Gunewardena S, Kumar TR.\u003c/strong\u003e Identification of follicle-stimulating hormone-responsive genes in Sertoli cells during early postnatal mouse testis development. \u003cem\u003eAndrology.\u003c/em\u003e 2023;11(5):860-871. doi:10.1111/andr.13459.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMirshafa A, Nazari M, Jahani D, Shaki F.\u003c/strong\u003e Size-dependent neurotoxicity of aluminum oxide nanoparticles: A comparison between nano- and micrometer-sized particles. \u003cem\u003eBiol Trace Elem Res.\u003c/em\u003e 2018;183(2):261-269. doi:10.1007/s12011-017-1142-8.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMansour FR, Nabiuni M, Amini E.\u003c/strong\u003e Ovarian toxicity induced by aluminum chloride: alteration of Cyp19a1, Pcna, Puma, and Map1lc3b gene expression. \u003cem\u003eToxicology.\u003c/em\u003e 2021;153084.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSong Y, Xue Y, Liu X, Wang P, Liu L.\u003c/strong\u003e Effects of acute exposure to aluminum on blood-brain barrier and the protection of zinc. \u003cem\u003eNeurosci Lett.\u003c/em\u003e 2008;445(1):42-6. doi: 10.1016/j.neulet.2008.08.081.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eCheraghi E, Golkar A, Roshanaei K, Alani B.\u003c/strong\u003e Aluminium-induced oxidative stress, apoptosis, and alterations in testicular tissue and sperm quality in Wistar rats: ameliorative effects of curcumin. \u003cem\u003eInt J Fertil Steril.\u003c/em\u003e 2017;11(3):166.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eThanaraj S, Mitthun ANK, Geetha Sravanthy P, Carmelin DS, Surya M, Saravanan M.\u003c/strong\u003e Green synthesis of aluminum oxide nanoparticles using \u003cem\u003eClerodendrum\u003c/em\u003e\u003cem\u003e\u0026nbsp;phlomidis\u003c/em\u003e and their antibacterial, anti-inflammatory, and antioxidant activities. \u003cem\u003eCureus.\u003c/em\u003e 2024;16(1):e52279. doi:10.7759/cureus.52279.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHuang YW, Wu CH, Aronstam RS.\u003c/strong\u003e Toxicity of transition metal oxide nanoparticles: recent insights from in vitro studies. \u003cem\u003eMaterials.\u003c/em\u003e 2010;3(10):4842-59.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMirshafa A, Nazari M, Jahani D, Shaki F.\u003c/strong\u003e Size-dependent neurotoxicity of aluminum oxide nanoparticles: a comparison between nano- and micrometer size on the basis of mitochondrial oxidative damage. \u003cem\u003eBiol Trace Elem Res.\u003c/em\u003e 2018;183(2):261-269. doi:10.1007/s12011-017-1142-8.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eArzuaga X, Smith MT, Gibbons CF, Skakkeb\u0026aelig;k NE, Yost EE, Beverly BEJ, et al.\u003c/strong\u003e Proposed key characteristics of male reproductive toxicants as an approach for organizing and evaluating mechanistic evidence in human health hazard assessments. \u003cem\u003eEnviron Health Perspect.\u003c/em\u003e 2019;127(6):65001. doi:10.1289/EHP5045.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHamdi H.\u003c/strong\u003e Testicular dysfunction induced by aluminum oxide nanoparticle administration in albino rats and the possible protective role of pumpkin seed oil. \u003cem\u003eJ Biol Anim Zool.\u003c/em\u003e 2020; 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1275:175-203. doi:10.1007/978-3-030-47492-7_8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alpha tocopherol succinate, Alumina nanoparticles, Sertoli cells, Inhibin B, Connexin 43, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7947162/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7947162/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSertoli cells are essential for spermatogenesis, controlling the microenvironment and interacting with germ cells. This study evaluates the preventive effect of alpha-tocopherol succinate against Al₂O₃-NPs-induced damage in NMRI mice Sertoli cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe effects of Al₂O₃-NPs and vitamin E were assessed on Sertoli cells using viability assays, oxidative stress markers, apoptosis detection, and gene expression analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAl₂O₃-NPs significantly reduced Sertoli cell viability and increased apoptosis, while vitamin E treatment mitigated these effects. Expression of INHBB and Cx43 was significantly downregulated by Al₂O₃-NPs and upregulated with vitamin E.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eVitamin E alleviates Al₂O₃-NPs-induced Sertoli cell damage by reducing oxidative stress, inhibiting apoptosis, and restoring INHBB and Cx43 expression.\u003c/p\u003e","manuscriptTitle":"Protective Effect of Alpha Tocopherol Succinate on Al₂O₃-NPs Induced Damage in NMRI Mice Sertoli Cells: The Role of Inhibin B and Connexin 43","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 10:51:54","doi":"10.21203/rs.3.rs-7947162/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-09T10:46:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T06:43:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234079109734766129148779419963385560210","date":"2025-11-25T15:33:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308468095594543180804474495400293073454","date":"2025-10-29T15:53:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164258784060082354501334605174145492759","date":"2025-10-29T12:50:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-29T12:28:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-28T11:36:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-28T11:35:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-10-25T13:53:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7013586f-ff78-46d4-931f-12ee85d1a30d","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:01:33+00:00","versionOfRecord":{"articleIdentity":"rs-7947162","link":"https://doi.org/10.1007/s11033-026-11566-8","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2026-03-03 15:57:01","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-11-10 10:51:54","video":"","vorDoi":"10.1007/s11033-026-11566-8","vorDoiUrl":"https://doi.org/10.1007/s11033-026-11566-8","workflowStages":[]},"version":"v1","identity":"rs-7947162","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7947162","identity":"rs-7947162","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}