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.

[Impact of 4-bromobiphenyl ether on the expression of γH2AX in the rat testis].

Objective: To investigate the effects of monobromobiphenyl ether (4-BDE) on the expression of γH2AX in the rat testis, and the possible mechanism of 4-BDE affecting the reproductive function of the male rats. METHODS: Twenty-four SD male rats were randomly divided into 4 groups: control and low-, medium- and high-dose 4-BDE, the control rats treated intragastrically with olive oil, and the animals in the latter three groups with 4-BDE at 50, 100 and 200 mg/kg/d, respectively, all for 30 consecutive days. Then all the rats were killed and the testis tissues harvested for HE staining, examination of the apoptosis of the cells by TUNEL and determination of the expressions of H2AX mRNA and γH2AX by q-PCR and Western blot. RESULTS: HE staining manifested occasional reduction or absence of spermatogonial and Sertoli cells in the seminiferous tubules in the medium- and high-dose 4-BDE groups. Compared with the controls, the rats exposed to 4-BDE showed a significant dose-dependent increase in the apoptosis of the testis tissue (P < 0.05), even more significant in the medium- and high-dose 4-BDE groups than in the low-dose group (P < 0.05). There was a dose-effect relationship in the apoptosis index, but with no statistically significant difference between the medium- and high-dose 4-BDE groups (P > 0.05). The results of q-PCR exhibited no statistically significant difference in the expression level of H2AX mRNA either between the control and 4-BDE-exposed rats (P > 0.05) or among the three 4-BDE groups (P > 0.05). The expression of the γH2AX protein was remarkably higher in the 4-BDE groups than in the control (P < 0.05), but not significantly different among the 4-BDE groups (P > 0.05). CONCLUSIONS: Exposure to 4-BDE at 100 or 200 mg/kg/d damages the structure of seminiferous tubules, increases the apoptosis of testicular cells, significantly up-regulates the expression of the γH2AX protein, and consequently increases DNA double-strand breaks (DSB) in the rat testis. The apoptosis of testicular cells may be related to DSB./.

[Protective effect of taurine against formaldehyde-induced male reproductive toxicity in adult male rats].

OBJECTIVE: To study the effect of taurine on the reproductive toxicity damage induced by formaldehyde (FM) in adult male rats. METHODS: Forty-eight SD adult male rats were equally randomized into a normal control, an FM poisoning (FMP), a taurine intervention (TI), and an TI+FMP group. The control rats were given normal diet and gavage of saline, the rats of the FMP group treated intraperitoneally with FM at 10 mg/kg qd alt, those of the TI group intragastrically with taurine at 100 mg/kg qd, and those of the TI+FMP group with both FM and taurine at the above doses. After 30 days of treatment, the blood of the abdominal cardinal vein of the rats was extracted for measurement of the levels of serum hormones, the body weight, testis weight and testicular coefficient obtained, the testis tissue subjected to HE staining, and the expressions of Bcl-2 and Bax determined by Western blot. RESULTS: There were no statistically significant differences among the four groups of rats in the body weight, testis weight or testicular coefficient (P > 0.05). The rats in the FMP group showed obviously decreased testicular spermatogenic cells and disordered layers and loose structure of seminiferous tubules, which were basically restored to normal after taurine intervention. Compared with the normal controls, the animals of the TI group exhibited no significant abnormality, but those of the FMP group presented markedly reduced levels of serum T, LH and FSH (P < 0.05), and dramatically increased level of Gonadotropin-releasing hormone (GnRH) (P < 0.01). The levels of serum hormones were all significantly improved (P < 0.05) and that of the apoptotic protein Bax basically returned to normal (P < 0.05) after taurine intervention. CONCLUSIONS: Taurine has a certain protective effect against male reproductive toxicity caused by formaldehyde.

[Fluorosis of coal burning affects the male reproductive system].

Fluorosis of coal burning is a new type of endemic fluorosis in China, which affects the male reproductive system. Furthermore, the content of fluoride in the semen, sperm mortality, sperm concentration and the incidence of infertility are higher in severe fluorosis areas than in mild- and non-fluorosis areas, so are the levels of serum follicle-stimulating hormone and luteinizing hormone. However, the levels of inhibin B, serum testosterone and estradiol show different degrees of reduction in severe fluorosis areas. Accordingly, fluorosis of coal burning, just like other endemic fluorosis, may affect the structure of male reproductive organs, the generation of sperm and reproductive endocrinology, resulting in the decline of men's reproductive ability.

N6-Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis.

BACKGROUND: The present study investigated the regulatory effects of N6-methyladenosine (m6A) methyltransferase like-3 (METTL3) in diabetes-induced testicular damage. METHODS: In vivo diabetic mice and high glucose (HG) treated GC-1 spg cells were established. The mRNA and protein expressions were determined by real-time quantitative polymerase chain reaction, Western blot, immunofluorescence and immunohistochemistry staining. Levels of testosterone, blood glucose, cell viability, and apoptosis were detected by enzyme-linked immunosorbent assay, MTT, and flow cytometry, respectively. Molecular interactions were verified by RNA immunoprecipitation and RNA pull-down assay. Histopathological staining was performed to evaluate testicular injury. RESULTS: METTL3 and long non-coding RNA taurine up-regulated 1 (lncRNA TUG1) were downregulated in testicular tissues of diabetic mice and HG-treated GC-1 spg cells. METTL3 overexpression could reduce the blood glucose level, oxidative stress and testicular damage but enhance testosterone secretion in diabetic mouse model and HG-stimulated GC-1 spg cells. Mechanically, METTL3-mediated m6A methylation enhanced the stability of TUG1, then stabilizing the clusterin mRNA via recruiting serine and arginine rich splicing factor 1. Moreover, inhibition of TUG1/clusterin signaling markedly reversed the protective impacts of METTL3 overexpression on HG-stimulated GC-1 spg cells. CONCLUSION: This study demonstrated that METTL3 ameliorated diabetes-induced testicular damage by upregulating the TUG1/clusterin signaling. These data further elucidate the potential regulatory mechanisms of m6A modification on diabetes-induced testicular injury.

[Effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis].

OBJECTIVE: To explore the effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis. METHODS: Totally 40 male SD rats were equally randomized into four groups control group, low fluorosis group, middle fluorosis group, and high fluorosis group. Rats in all three fluorosis groups were fed with corn dried by burning coal obtained from endemic fluorosis areas with high fluoride, and thus the animal models of fluorosis were established. After 120 and 180 days, all the rats were sacrificed. Testis tissues were stained with hematoxylin eosin and observed under light microscope. The malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, total nitric oxide synthase (TNOS), and inducible nitric oxidase synthase (iNOS) were measured by biochemical methods in the testis tissues. The content of NaF in testis was measured by fluorine selective electrode. RESULTS: The rat fluorosis models were successfully established. The fluoride content in testis was significantly increased in all the fluorosis groups(P<0.01). Testicular structures were damaged in all of fluoride groups. The TNOS, iNOS activities, and MDA content of each fluoride group were significantly higher than that of the control group on day 120 and 180 (P<0.05 or 0.01 ). The TNOS, iNOS activities, and MDA content significantly increased in a dose dependent manner (P<0.05 or 0.01). The SOD activities significantly decreased in all the fluoride groups (P<0.05 or 0.01). CONCLUSIONS: Endemic fluoride poisoning caused by coal burning can cause disorders in the oxidative system and antioxidative system in rat testis. The oxidative stress may play an important role in the fluorides induced reproductive toxicity in male rats.