BRD7 facilitates ferroptosis via modulating clusterin promoter hypermethylation and suppressing AMPK signaling in diabetes-induced testicular damage.

BACKGROUND: Diabetes mellitus (DM)-induced testicular damage is associated with sexual dysfunction and male infertility in DM patients. However, the pathogenesis of DM-induced testicular damage remains largely undefined. METHODS: A streptozotocin (STZ)-induced diabetic model and high glucose (HG)-treated in vitro diabetic model were established. The histological changes of testes were assessed by H&E staining. Serum testosterone, iron, MDA and GSH levels were detected using commercial kits. Cell viability and lipid peroxidation was monitored by MTT assay and BODIPY 581/591 C11 staining, respectively. qRT-PCR, immunohistochemistry (IHC) or Western blotting were employed to detect the levels of BRD7, Clusterin, EZH2 and AMPK signaling molecules. The associations among BRD7, EZH2 and DNMT3a were detected by co-IP, and the transcriptional regulation of Clusterin was monitored by methylation-specific PCR (MSP) and ChIP assay. RESULTS: Ferroptosis was associated with DM-induced testicular damage in STZ mice and HG-treated GC-1spg cells, and this was accompanied with the upregulation of BRD7. Knockdown of BRD7 suppressed HG-induced ferroptosis, as well as HG-induced Clusterin promoter methylation and HG-inactivated AMPK signaling in GC-1spg cells. Mechanistical studies revealed that BRD7 directly bound to EZH2 and regulated Clusterin promoter methylation via recruiting DNMT3a. Knockdown of Clusterin or inactivation of AMPK signaling reverses BRD7 silencing-suppressed ferroptosis in GC-1spg cells. In vivo findings showed that lack of BRD7 protected against diabetes-induced testicular damage and ferroptosis via increasing Clusterin expression and activating AMPK signaling. CONCLUSION: BRD7 suppressed Clusterin expression via modulating Clusterin promoter hypermethylation in an EZH2 dependent manner, thereby suppressing AMPK signaling to facilitate ferroptosis and induce diabetes-associated testicular damage.

Semaglutide mitigates testicular damage in diabetes by inhibiting ferroptosis.

Diabetes is linked to male infertility, but the mechanisms and therapeutic options remain unclear. This study investigates the effects of semaglutide on testicular function in a diabetes mouse model. Clinical data shows that diabetes affects blood glucose, lipid levels, and sperm quality. Single-cell and transcriptome analyses reveal changes in testicular tissue cell proportions and activation of ferroptosis pathways in diabetic patients/rats. In the diabetes mouse model, sperm quality decreases significantly. Treatment with semaglutide (Sem) and the ferroptosis inhibitor ferrostatin-1 (Fer-1) alleviates testicular damage, as evidenced by improved lipid peroxidation and ferroptosis markers. Moreover, the diabetes-induced decrease in the TM-3 cell line's vitality, increased lipid peroxidation, ROS, ferrous ions, and mitochondrial membrane potential damage are all improved by semaglutide and ferrostatin-1 intervention. Overall, these findings highlight semaglutide's potential as a therapeutic approach for mitigating diabetes-induced testicular damage through modulation of the ferroptosis pathway.

A novel approach of using Maca root as a radioprotector in a rat testicular damage model focusing on GRP78/CHOP/Caspase-3 pathway.

PURPOSE: Despite the effectiveness of ionizing radiation in treating cancer, it can damage healthy tissues in the vicinity. Due to the high radio-sensitivity of testicular tissues, radiation therapy may affect spermatogenesis, which may result in infertility. Hence, in this study testicular damage model is constructed to investigate the mitigation effect of Maca root powder and its potential radioprotective activity through both oxidative and endoplasmic reticulum (ER) stresses, besides the apoptotic pathway. METHODS: Male albino rats were exposed to 6Gy of whole-body gamma radiation single dose. Maca root powder (1 g/kg b.wt./day, by oral gavage) was administered for a week before irradiation, then d-galactose (300 mg/kg, by oral gavage) and Maca daily for another week. RESULTS: Gamma radiation and d-galactose revealed a significant decrease in serum testosterone, sperm count, and motility and higher percentage of the sperm head abnormality, while Maca root treatment maintained all sperm morphology parameters. Maca root treatment demonstrated a notable defense against radiation-induced oxidative stress and ameliorated malonaldehyde (MDA), reactive oxygen species (ROS), nitric oxide (NO), glutathione-S-transferase (GST) levels, reduced glutathione (GSH), oxidized glutathione (GSSG) and the ratio of GSH/GSSG in testis tissues. Exposure to gamma rays and d-galactose displayed a significant elevation in GRP78, CHOP, total caspase-3 as well as active (cleaved) caspase-3 levels, whereas treatment with Maca significantly reduced the ER and apoptotic markers levels. Also, Maca improved the histological changes of the disorganized seminiferous tubules induced by irradiation. CONCLUSION: Our findings show for the first time that Maca has a protective effect on male reproductive damage induced by radiotherapy. Maca root reveals anti-apoptotic effect and protection against testicular damage via GRP78/CHOP/caspase-3 pathway.

Diquat causes mouse testis injury through inducing heme oxygenase-1-mediated ferroptosis in spermatogonia.

Diquat dibromide (DQ) is a globally used herbicide in agriculture, and its overuse poses an important public health issue, including male reproductive toxicity in mammals. However, the effects and molecular mechanisms of DQ on testes are limited. In vivo experiments, mice were intraperitoneally injected with 8 or 10 mg/kg/ day of DQ for 28 days. It has been found that heme oxygenase-1 (HO-1) mediates DQ-induced ferroptosis in mouse spermatogonia, thereby damaging testicular development and spermatogenesis. Histopathologically, we found that DQ exposure caused seminiferous tubule disorders, reduced germ cells, and increased sperm malformation, in mice. Reactive oxygen species (ROS) staining of frozen section and transmission electron microscopy (TEM) displayed DQ promoted ROS generation and mitochondrial morphology alterations in mouse testes, suggesting that DQ treatment induced testicular oxidative stress. Subsequent RNA-sequencing further showed that DQ treatment might trigger ferroptosis pathway, attributed to disturbed glutathione metabolism and iron homeostasis in spermatogonia cells in vitro. Consistently, results of western blotting, measurements of MDA and ferrous iron, and ROS staining confirmed that DQ increased oxidative stress and lipid peroxidation, and accelerated ferrous iron accumulation both in vitro and in vivo. Moreover, inhibition of ferroptosis by deferoxamine (DFO) markedly ameliorated DQ-induced cell death and dysfunction. By RNA-sequencing, we found that the expression of HO-1 was significantly upregulated in DQ-treated spermatogonia, while ZnPP (a specific inhibitor of HO-1) blocked spermatogonia ferroptosis by balancing intracellular iron homeostasis. In mice, administration of the ferroptosis inhibitor ferrostatin-1 effectively restored the increase of HO-1 levels in the spermatogonia, prevented spermatogonia death, and alleviated the spermatogenesis disorders induced by DQ. Overall, these findings suggest that HO-1 mediates DQ-induced spermatogonia ferroptosis in mouse testes, and targeting HO-1 may be an effective protective strategy against male reproductive disorders induced by pesticides in agriculture.

Beneficial effects of adipose-derived stromal vascular fraction on testicular injury caused by busulfan.

The use of stem cells can attenuate testicular injury and promote sperm production. The adipose-derived stromal vascular fraction (SVF) has become an attractive cell source for cell-based therapies. In this study, we aimed to investigate the therapeutic efficacy of SVF on busulfan-induced testicular damage in rats. Twenty-four male rats were randomly divided into control, busulfan, SVF, and busulfan + SVF groups. Testicular damage was induced by intraperitoneal administration of busulfan (35 mg/kg). SVF obtained from human adipose tissue using Lipocube SVF™ was injected into rats 5 weeks after busulfan administration. At the end of the 8th week, rats were sacrificed, and histopathological, biochemical, and western blotting analyses were performed. No harmful effects of SVF on healthy testis tissue and sperm parameters were detected. SVF improved busulfan-induced oxidative stress in both testis tissue and serum. SVF injection to damaged testicular tissue resulted in increases in the healthy spermatozoon numbers and decreases in the abnormal tail numbers. Additionally, SVF increased bax/Bcl, DAZL, and TGF-ß1 levels whereas decreased ATG5 and NF-kB levels. According to the results we obtained in this study, we suggest that SVF is beneficial in restoring damaged tissue by primarily being a multipotent cell source, by inhibiting oxidative stress and converting necrotic cell death to apoptotic cell death. In the future, clinical applications should bring higher benefits. Since SVF is the patient's own tissue, being harmless, it will offer an advantageous supportive treatment option for patients already weakened by cancer and anticancer therapy.

Di-(2-ethylhexyl) phthalate induces ferroptosis in prepubertal mouse testes via the lipid metabolism pathway.

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, has been shown to cause reproductive toxicity, but the precise mechanism remains unclear. This study aimed to investigate the possible molecular mechanism of DEHP-induced testicular damage. In vivo study, we administered different doses of DEHP (0, 250, and 500 mg/kg/day) to male C57BL/6 mice from 22 and 35 days after birth. We found that DEHP exposure induced histopathological alterations in prepubertal testes, and testicular lipidomics indicated notable alterations in lipid metabolism and significant enrichment of ferroptosis. Further tests showed that ferrous iron (Fe2+ ) and malondialdehyde (MDA) levels significantly increased after DEHP exposure. Western blotting revealed that DEHP exposure reduced glutathione peroxidase 4 (GPX4) expression, and elevated acyl coenzyme A synthetase long-chain member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) expression. The in vitro results were consistent with the in vivo results. When Leydig cells and Sertoli cells were treated with ferrostatin-1 and monoethylhexyl phthalate (MEHP), MEHP-induced increases in Fe2+ and MDA levels, accumulation of lipid reactive oxygen species, downregulation of GPX4, and upregulation of ACSL4 and LPCAT3 were reversed. Collectively, our findings suggested that aberrant lipid metabolism and ferroptosis may be involved in prepubertal DEHP exposure-induced testicular damage.

Arginine Biosynthesis Mediates Wulingzhi Extract Resistance to Busulfan-Induced Male Reproductive Toxicity.

Busulfan, an indispensable medicine in cancer treatment, can cause serious reproductive system damage to males as a side effect of its otherwise excellent therapeutic results. Its widespread use has also caused its accumulation in the environment and subsequent ecotoxicology effects. As a Chinese medicine, Wulingzhi (WLZ) has the effects of promoting blood circulation and improving female reproductive function. However, the potential effects of WLZ in male reproduction and in counteracting busulfan-induced testis damage, as well as its probable mechanisms, are still ambiguous. In this study, busulfan was introduced in a mouse model to evaluate its production of the testicular damage. The components of different WLZ extracts were compared using an untargeted metabolome to select extracts with greater efficacy, which were further confirmed in vivo. Here, we demonstrate abnormal spermatogenesis and low sperm quality in busulfan-injured testes. The WLZ extracts showed a strong potential to rehabilitate the male reproductive system; this effect was more prominent in room-temperature extracts. Additionally, both water and ethanol WLZ extracts at room temperature alleviated various busulfan-induced adverse effects. In particular, WLZ recovered spermatogenesis, re-activated arginine biosynthesis, and alleviated the increased oxidative stress and inflammation in the testis, ultimately reversing the busulfan-induced testicular injury. Collectively, these results suggest a promising approach to protecting the male reproductive system from busulfan-induced adverse side effects, as well as those of other similar anti-cancer drugs.

Protective roles of some natural and synthetic aromatase inhibitors in testicular insufficiency caused by Bisphenol A exposure.

In our study, the protective role of synthetic aromatase inhibitors anastrozole (ANS), letrozole (LTZ) and exemestane (EXM) and natural aromatase inhibitors resveratrol (RSV) and apigenin (APG) against testicular failure caused by exposure to Bisphenol A (BPA) was investigated. The epididymal sperm concentration, sperm motility and sperm morphology were determined. Oxidative stress and inflammatory response parameters were examined and histological examinations were performed in testicular tissues. Our results revealed that BPA exposure decreased serum testosterone and estrogen levels, increased FSH and LH levels (p < 0.05). BPA has been found to increase oxidative stress and inflammatory response and disrupt the histological structure. Also, BPA exposure decreased testicular weight, epididymal sperm concentration and motility, and increased abnormal sperm rate (p < 0.05). These results show that ANS, LTZ and RSV treatments reduce the BPA-induced testicular damage.

Attenuative effect of astilbin on polystyrene microplastics induced testicular damage: Biochemical, spermatological and histopathological-based evidences.

Polystyrene microplastics (PS-MPs) are the potential environmental pollutants that possess the ability to induce testicular damage. Astilbin (ASB) is a dihydroflavonol, abundantly reported in multiple plants that has various pharmacological properties. This research elucidated the mitigative potential of ASB against PS-MPs-instigated testicular toxicity. 48 adult male rats (200 ± 10 g) were distributed into 4 groups (n = 12): control, PS-MPs received (0.01 mg/kg), PS-MPs + ASB received (0.01 mg/kg + 20 mg/kg) and ASB supplemented group (20 mg/kg). After 56th day of the trial, animals were sacrificed and testes were harvested for the estimation of biochemical, hormonal, spermatogenic, steroidogenic, apoptotic and histological profiles. PS-MPs intoxication significantly (P < 0.05) lowered glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GSR) as well as catalase (CAT) activities, whereas elevated MDA as well as ROS levels. Besides, the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), nuclear factor kappa-B (NF-κB) along with cyclooxygenase-2 (COX-2) activity were raised. PS-MPs treatment reduced luteinizing hormone (LH), plasma testosterone and follicle-stimulating hormone (FSH) level besides decreased epididymal sperm number, viability, motility as well as the count of HOS coil-tailed spermatozoa and increased sperm morphological irregularities. PS-MPs exposure lowered steroidogenic enzymes (17ß-HSD, 3ß-HSD and StAR protein along with Bcl-2 expression, besides increasing Caspase-3 and Bax expressions and histopathological alterations in testicular tissues. However, ASB treatment significantly reversed PS-MPs mediated damage. In conclusion, ASB administration is protective against PS-MPs-instigated testicular damage owing to its anti-inflammatory, anti-apoptotic, antioxidant and androgenic nature.

Antioxidant, anti-inflammatory, and anti-apoptotic effects of genkwanin against aflatoxin B1-induced testicular toxicity.

Aflatoxin B1 (AFB1) is the most hazardous aflatoxin that causes significant damage to the male reproductive system. Genkwanin (GNK) is a bioactive flavonoid that shows antioxidant and anti-inflammatory potential. Therefore, the current study was planned to evaluate the effects of GNK against AFB1-induced testicular toxicity. Forty-eight male rats were distributed into four groups (n = 12 rats). AFB1 (50 µg/kg) and GNK (20 mg/kg) were administered to the rats for eight weeks. Results of the current study revealed that AFB1 exposure induced adverse effects on the Nrf2/Keap1 pathway and reduced the expressions and activities of antioxidant enzymes. Additionally, it increased the levels of oxidative stress markers. Furthermore, expressions of steroidogenic enzymes were down-regulated by AFB1 intoxication. Besides, AFB1 exposure reduced the levels of gonadotropins and plasma testosterone, which subsequently reduced the epididymal sperm count, motility, and hypo-osmotic swelled (HOS) sperms, while increasing the number of dead sperms and causing morphological anomalies of the head, midpiece, and tail of the sperms. In addition, AFB1 decreased the activities of testicular function marker enzymes and the levels of inflammatory markers. Moreover, it severely affected the apoptotic profile by up-regulating the expressions of Bax and Casp3, while down-regulating the Bcl2 expression. Besides, AFB1 significantly damaged the histoarchitecture of testicular tissues. However, GNK treatment reversed all the AFB1-induced damages in the rats. Taken together, the current study reports the potential use of GNK as a therapeutic agent to prevent AFB1-induced testicular toxicity due to its antioxidant, anti-inflammatory, and anti-apoptotic properties.

CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

Role of EGF/ERK1/2/HO-1 axis in mediating methotrexate induced testicular damage in rats and the ameliorative effect of xanthine oxidase inhibitors.

Objectives: Methotrexate (MTX) is commonly used in the management of several malignancies and autoimmune disorders; however, testicular damage is one of the most detrimental effects of MTX administration. The current the protective effect of xanthine oxidase inhibitors either purine analogue; allopurinol (ALL) or non-purine analogue; febuxostat (FEB) in testicular injury induced by MTX in rats.Materials and methods: Thirty-two rats were randomly allocated to four groups; control (received vehicles), MTX (received single dose, 20 mg/kg, i.p.), MTX + ALL (received MTX plus ALL) and MTX + FEB (received MTX plus ALL). ALL and FEB were administered orally at 100- and 10 mg/kg, respectively for 15 days. Total and free testosterone were measured in serum. In addition, total antioxidant capacity (TAC), epidermal growth factor (EGF), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), extracellular signal-regulating kinase1/2 (ERK1/2), and total nitrite/nitrate (NOx) end products were measured in testicular tissues. At the same time, immunoexpression of HO-1in testicular tissue was measured. Histopathological examination was done.Results: ALL and FEB increased total and free serum testosterone. Both drugs showed a significant reduction in testicular MDA, NOx, TNF-α levels with an increase in TAC, EGF, and ERK1/2 levels in testicular tissue. Furthermore, both drugs enhanced HO-1 immunoexpression in testicular tissue. All these findings were parallel to the preservation of normal testicular architecture in rats treated with ALL and FEB.Conclusion: All and FEB were equally protective against testicular damage induced by MTX through anti-inflammatory, anti-apoptotic, and antioxidant actions. Their effects might be through activation of the EGF/ERK1/2/HO-1 pathway.

The protective effect of crocin against testicular toxicity induced by ionizing radiation via AKT/FOXO pathway.

Crocin, a pharmacologically active component of Crocus sativus L. (saffron), has been informed to be beneficial in the treatment of stress-related oxidative impairment. In the present study, we examined the protective role of crocin against testicular damage induced by radiation (acute and fractionated) and the alteration of the AKT/FOXO signaling pathway. Male Wister albino rats were exposed to acute dose of 6 Gy and a fractionated dose of gamma radiation (2 Gy every 2 days up to 6 Gy total doses). Rats were pretreated intraperitoneally with crocin in a dose of 50 mg/kg for seven consecutive days prior to exposure to irradiation at a level of 6 Gy and during the fractionated irradiation of rats. Control groups were run concurrently. Ionizing radiation caused changes in the level of oxidative stress biomarkers manifested as elevation of thiobarbituric acid reactive substance, total nitrate/nitrite and reactive oxygen species (ROS) associated with a decrease in catalase as well as in the level of inflammatory parameters (decrease in expression of Nrf2 which was related to a significant increase in expression of NF-κB p65). Irradiation produced cellular damage characterized by an increase in serum lactate dehydrogenase. These findings were aligned with increased expression of the forkhead box O-1 (FOXO-1) and activation of protein kinase B (AKT) pathway. Irradiation of rats led to reduction in serum testosterone level and testicular weights. Pretreatment with the indicated dose of crocin shielded against the changes in all the evaluated parameters. Administration of crocin can be introduced as a novel preclinical approach for regulation of testicular damage induced by radiation; via controlling the ongoing oxidative stress and inflammatory reaction as well as activation FOXO/AKT signaling pathway.

Mechanistic Protective Effect of Cilostazol in Cisplatin-Induced Testicular Damage via Regulation of Oxidative Stress and TNF-α/NF-κB/Caspase-3 Pathways.

Despite being a potent anticancer drug, cisplatin has limited applicability due to its adverse effects, such as testicular damage. Consequently, reducing its toxicity becomes necessary. In this study, a selective phosphodiesterase-3 inhibitor, cilostazol, which is used to treat intermittent claudication, was examined for its ability to abrogate cisplatin-induced testicular toxicity. Its ameliorative effect was compared to that of two phosphodiesterase inhibitors, tadalafil and pentoxifylline. The study also focused on the possible mechanisms involved in the proposed protective effect. Cisplatin-treated rats showed a significant decrease in sperm number and motility, serum testosterone, and testicular glutathione levels, as well as a significant elevation in malondialdehyde, total nitrite levels, and the protein expression of tumor necrosis factor-alpha, nuclear factor-kappa ß, and caspase-3. These outcomes were confirmed by marked testicular architecture deterioration. Contrary to this, cilostazol, in a dose-dependent manner, showed potential protection against testicular toxicity, reversed the disrupted testicular function, and improved histological alterations through rebalancing of oxidative stress, inflammation, and apoptosis. In addition, cilostazol exerted a more pronounced protective effect in comparison to tadalafil and pentoxifylline. In conclusion, cilostazol ameliorates cisplatin-induced testicular impairment through alteration of oxidative stress, inflammation, and apoptotic pathways, offering a promising treatment for cisplatin-induced testicular damage.

Chromium Picolinate Protects against Testicular Damage in STZ-Induced Diabetic Rats via Anti-Inflammation, Anti-Oxidation, Inhibiting Apoptosis, and Regulating the TGF-ß1/Smad Pathway.

Chromium picolinate (CP) is an organic compound that has long been used to treat diabetes. Our previous studies found CP could relieve diabetic nephropathy. Thus, we speculate that it might have a positive effect on diabetic testicular injury. In this study, a diabetic rat model was established, and then the rats were treated with CP for 8 weeks. We found that the levels of blood glucose, food, and water intake were reduced, and body weight was enhanced in diabetic rats after CP supplementation. Meanwhile, in CP treatment groups, the levels of male hormone and sperm parameters were improved, the pathological structure of the testicular tissue was repaired, and testicular fibrosis was inhibited. In addition, CP reduced the levels of serum inflammatory cytokines, and decreased oxidative stress and apoptosis in the testicular tissue. In conclusion, CP could ameliorate testicular damage in diabetic rats, as well as being a potential testicle-protective nutrient in the future to prevent the testicular damage caused by diabetes.

[Ferroptosis is involved in testicular injury induced by TDCIPP in adolescent male mice].

Objective: To investigate the role of ferroptosis in testicular injury in adolescent male mice induced by TDCIPP. Methods: In December 2021, 30 healthy 3-week-old male C57BL/6 mice, with a body weight of (13±2) g, were selected and fed adaptive for one week. They were divided into control group, low-dose group, medium-dose group, high-dose group and iron death inhibitor group according to a random number table, with 6 mice in each group. Mice in low, medium and high dose groups were treated with 5, 25 and 125 mg/ (kg·d) TDCIPP for 28 days, respectively, while the control group was treated with the same amount of corn oil for 28 days. The iron death inhibitor group was given 125 mg/ (kg·d) TDCIPP intragastric administration for 28 days, and 30 mg/kg DFO saline solution was intraperitoneally injected three times a week. After the treatment, the mice were killed, the epididymis was separated, and sperm count was performed. HE staining was used to observe the morphological changes of mouse testis, and iron content in testis was detected by tissue iron detection kit. The level of reactive cxygen species, MDA content, and the mitochondrial membrane potential level of mice were detected. Western blot analysis of testicular glutathione peroxidase (GPX4) and internal cyclooxygenase-2 (COX2) protein expression. Results: Compared with the control group, the spermatogenic cells in the testes of mice treated with medium-and high-dose of TDCIPP were disorderly arranged, showing a vacuolar structure. the number of sperm in the epididymis was significantly reduced (P=0.009, 0.004), while the sperm deformity rate was significantly increased (P=0.010, 0.000). Moreover, the content of ROS, iron ion and MDA in the testes increased significantly (P<0.05), and the mitochondrial membrane potential of mouse testicular cells decreased significantly (P<0.05). The expression of GPX4 proteins decreased (P<0.05). while the expression of COX2 increased significantly (P<0.01). Compared with high-dose group group, spermatogenic cells in ferroptosis inhibitor group were closely arranged and normal, and ROS and Fe contents in testicular tissue were significantly decreased (P<0.01) ; GPX4 protein expression was significantly increased while COX2 protein expression was significantly decreased (P<0.05) . Conclusion: Ferroptosis is involved in TDCIPP-induced testicular damage in male pubertal mice.

Clusterin suppresses spermatogenic cell apoptosis to alleviate diabetes-induced testicular damage by inhibiting autophagy via the PI3K/AKT/mTOR axis.

BACKGROUND INFORMATION: Diabetes-induced testicular dysfunction is characterised by abnormal apoptosis of spermatogenic cells, but the underlying mechanism is poorly understood. This study aimed to investigate the roles of clusterin (CLU) in testicular damage associated with diabetes pathogenesis, as well as the molecular mechanism. A rat diabetes model was established using streptozocin, and the mouse spermatogenic cell line GC-1 spg was treated with high glucose as a cellular model. CLU was overexpressed in GC-1 spg cells, followed by detection of serum testosterone, cell proliferation, cell apoptosis and autophagy. RESULTS: CLU expression was significantly reduced and LC3 expression was elevated in testis tissues in the rat diabetes model and high glucose-treated GC-1 spg cells. High glucose led to suppressed viability, enhanced apoptosis, reduced Bcl-2 expression, elevated Bax expression and cleavage of Caspase-3/-9 in GC-1 spg cells, and these effects were abrogated by CLU overexpression. Additionally, CLU overexpression repressed LC3 and Beclin-1 expression, reduced the LC3II/LC3I ratio and promoted p62 expression in GC-1 spg cells in the presence of high glucose, and these effects were all mitigated by rapamycin treatment. Inhibition of PI3K/AKT/mTOR signalling with LY294002 activated autophagy in CLU-overexpressing GC-1 spg cells under high glucose conditions. CLU overexpression repressed autophagy and alleviated testicular damage in diabetic rats, which was also abrogated by LY294002 treatment. CONCLUSIONS: CLU expression is suppressed during diabetes-induced testicular damage, whereas CLU overexpression alleviates diabetes-induced testicular damage by activating PI3K/AKT/mTOR signalling to inhibit autophagy and further repress spermatogenic cell apoptosis.

Natural products against cisplatin-induced male reproductive toxicity: A comprehensive review.

Cisplatin is widely used as one of the most effective anticancer agents in the treatment of some neoplasms. Reproductive toxicity is the most common outcome associated with cisplatin testicular damage. Alternative natural medicines for treating male testicular disorders and infertility have received extensive attention in research. Natural products, medicinal herbs, and their secondary metabolites have been shown as promising agents in the management of testicular damage induced by chemotherapy drugs. This study aimed to review the research related to natural substances that are promising in mitigation of the cisplatin-induced toxicity in the reproductive system. PubMed and Scopus were searched for studies on various natural products for their potential protective property against reproductive toxicity induced by cisplatin from 2000 to 2020. Eligibility was checked based on selection criteria. Fifty-nine articles were included in this review. Mainly in animal studies, several natural agents have positively affected cisplatin-reproductive-toxicity factors, including reactive oxygen species, inflammatory mediators, DNA damage, and activation of the mitochondrial apoptotic pathway. Most of the natural agents were investigated in short-term duration and high doses of cisplatin exposure, considering their antioxidant activity against oxidative stress. Considering antioxidant properties, various natural products might be effective for the management of cisplatin reproductive toxicity. However, long-term recovery of spermatogenesis and management of low-dose-cisplatin toxicity should be considered as well as the bioavailability of these agents before and after treatment with cisplatin without affecting its anticancer activity.

The protective effect of metformin against testicular damage in diabetes and prostate cancer model.

Individuals with diabetes have an increased risk of breast, colorectal, pancreatic and prostate cancer. Metformin, an oral biguanide used to treat diabetes, has anti-hyperglycaemic, anti-hyperinsulinemic and antioxidant activities. The effects of metformin on testicular tissue damage in cancer and diabetic + cancer rat models were evaluated histologically, immunohistochemically and biochemically. The diabetic model was produced in Copenhagen rats using a single dose of streptozotocin (65 mg/kg), while prostate cancer was induced through subcutaneous inoculation of 2 × 104 Mat-LyLu cells into the animals. At the end of the experimental period, testicular tissues with a close functional relationship to the prostate were collected. Histological evaluation found moderate to severe damage to testes following the diabetes and cancer process. Histopathological and biochemical impairments were observed in the early stage of prostate cancer, which were increased in the diabetic animals. Metformin administration reversed these injuries and provided substantial protection of the testes. In particular, metformin had protective effects on tissue damage, apoptosis, oxidative stress and antioxidant capacity. This suggests that metformin should be further investigated as a targeted protective drug against prostate cancer-related damage to the testes.

miR-27b-3p Improved High Glucose-Induced Spermatogenic Cell Damage via Regulating Gfpt1/HBP Signaling.

INTRODUCTION: Diabetes mellitus (DM)-induced testicular damage is characterized by abnormal apoptosis of spermatogenic cells. Here, we clarified the roles and the molecular mechanism of microRNA (miR)-27b-3p in high glucose (HG)-induced spermatogenic cell damage. METHODS: GC-1 spg cells were treated with 30 mmol/L glucose for 24 h. Cell viability was assessed by 2.3 3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay. And, levels of O-linked N-acetylglucosamine (OGT), apoptosis-related proteins, and autophagy-related proteins were evaluated using Western blot. Levels of tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, and UDP-N-acetylglucosamine (UDP-GlcNAc) were assessed by enzyme linked immunosorbent (ELISA) assay. Levels of reactive oxygen species (ROS), malonic dialdehyde (MDA) and activity of superoxide dismutase (SOD) in cells were determined using kits. Cell apoptosis was determined using flow cytometry assay. Besides, dual luciferase reporter assay was employed to verify the binding relationship between miR-27b-3p and glutamine-fructose-6-phosphate transaminase 1 (Gfpt1). RESULTS: miR-27b-3p was markedly downregulated in HG-treated GC-1 spg cells. HG treatment caused decreased cell viability, increased oxidative stress and inflammation, and induced autophagy and apoptosis, which were abolished by miR-27b-3p overexpression. miR-27b-3p suppressed the activation of hexosamine biosynthetic pathway (HBP) signaling in HG-treated spermatogenic cells. miR-27b-3p directly bound to Gfpt1 and negatively regulated its expression. CONCLUSION: miR-27b-3p could improve HG-induced spermatogenic cell damage via regulating Gfpt1/HBP signaling, providing a new treatment strategy for the treatment of DM-induced testicular damage.