Effects of Zn deficiency,antioxidants, and low-dose radiation on diabetic oxidative damage and cell death in the testis

Infertility is one of the common complications in diabetic men and mainly due to the loss of germ cells by apoptotic cell death. Although several mechanisms have been proposed to explain the induction of testicular cell death by diabetes, diabetic induction of testicular oxidative stress and damage may be the predominant mechanism responsible for the testicular cell death in diabetes. To explore whether factors that either increase or decrease the testicular oxidative stress and damage will enhance or prevent diabetes-induced testicular cell death, the effect of zinc (Zn) deficiency on diabetes-induced cell death has been examined since Zn was found to play an important role in the protection of testis from oxidative stress and damage. Zn deficiency, induced by its chelator N,N,N,N-Tetrakis(2-pyridylmethyl)-1,2-ethylenediamine, was found to exacerbate diabetes-induced testicular oxidative damage and cell death. In contrast, treatment of diabetic rats with antioxidant N-acetylcysteine or low-dose radiation that can up-regulate endogenous antioxidants significantly attenuated diabetes-induced testicular cell death. These results suggest that diabetes-induced testicular cell death that may eventually cause men’s infertility is predominantly mediated by the oxidative stress and damage. To prevent or delay diabetes-caused infertility, diabetic patients should avoid Zn deficiency, and might consider antioxidant supplementation.     

Repetitive exposures to low-dose X-rays attenuate testicular apoptotic cell death in streptozotocin-induced diabetes rats

To define whether repetitive exposures to low-dose radiation (LDR) can attenuate diabetes-induced testicular cell death, Type 1 diabetic rats were produced by single injection of streptozotocin (STZ). Once hyperglycemia was diagnosed, diabetic rats were treated with and without LDR (25 and 50 mGy X-rays) daily for 4 weeks. Eight and 12 weeks after diabetes onset, testicular apoptotic cell death was examined by flow cytometry with Annexin V/PI staining, Western blotting assay for caspase-3 cleavage, and TUNEL staining for localization of apoptotic cells. Diabetes induced a significant increase in testicular apoptotic cell death, which was able to be attenuated by repetitive exposures to LDR. Diabetes-induced testicular cell death was associated with increased mitochondrial dysfunction, shown by the decreased mitochondrial potential and increased expressions of Bax mRNA and protein. All these changes were significantly attenuated in certain extends by repetitive exposures to LDR. To investigate the mechanisms by which LDR attenuates diabetes-induced testicular apoptotic cell death, serum sex hormone (testosterone, luteinizing hormone and follicle stimulating hormone) levels, and both serum and testicular oxidative damage (lipid peroxides) and antioxidant contents (superoxide dismutase, catalase and glutathione) were measured. Serum sex hormones were significantly decreased in diabetic rats, but not significantly in diabetic rats with multiple exposures to LDR; serum and testicular oxidative damage was significantly increased along with significant decreases in serum and testicular antioxidants in diabetic rats; however, these changes were significantly prevented by repetitive exposures to LDR. Furthermore, diabetic effects on the testicular oxidative damage and cell death were all attenuated by antioxidant N-acetylcysteine. These results suggest that diabetes-induced testicular cell death is probably mediated by increased oxidative stress. LDR protection from diabetes-induced testicular cell death is most likely mediated by its preserving antioxidants.     

Exacerbation of diabetes-induced testicular apoptosis by zinc deficiency is most likely associated with oxidative stress, p38 MAPK activation, and p53 activation in mice

Since diabetes induces testicular oxidative damage and cell death, and zinc (Zn) plays an important role in the spermatogenesis, the objective of the present study was to define the effects of Zn deficiency on diabetes-induced testicular apoptosis and associated mechanisms. Zn deficiency was induced by chronic treatment of normal and diabetic mice with N,N,N',N'-tetrakis (2-pyridylemethyl) ethylenediamine (TPEN) chelation. After diabetes onset, mice were given intraperitoneally TPEN at 5mg/kg daily for four months, which, like diabetes, induced a significant decrease in testicular Zn level. TUNEL staining revealed that testicular apoptosis was significantly increased along with an increased Bax/Bcl-2 ratio, in diabetic mice and TPEN-treated normal mice. Zn deficiency significantly exacerbated diabetes-induced testicular apoptosis, along with significantly increased oxidative and nitrosative damage and down-regulation of antioxidant Nrf2 expression. Increased oxidative stress was associated with an increase in activation of p38 MAPK and p53 protein in diabetic testis, which was worsened in the testes of diabetic mice with Zn deficiency. Diabetes also induced a significant increase in endoplasmic reticulum stress and associated cell death, which was not affected by Zn deficiency. These results suggest that like diabetes, chronic depletion of Zn with TPEN induces testicular oxidative stress and damage, along with the activation of p38 MAPK and p53 signaling and mitochondria-related apoptotic cell death. Therefore, prevention of Zn deficiency for diabetic patients is important in order to avoid the exacerbation of diabetic effects on testicular cells death.     

Protective effect of FGF21 on type 1 diabetes-induced testicular apoptotic cell death probably via both mitochondrial- and endoplasmic reticulum stress-dependent pathways in the mouse model

Fibroblast growth factor 21 (FGF21) is a novel member identified and was reported to express predominantly in pancreas, liver and adipose tissue, and relatively less in other organs, such as the testis. However, the role of FGF21 in the testis has never been addressed. The present study examined FGF21 expression at mRNA level by real-time RT-PCR assay in the testis of fasting and non-fasting mice or mice with type 1 diabetes that was induced with streptozotocin. We also examined the effect of Fgf21 gene deletion or supplementation of the exogenous FGF21 on the testicular apoptotic cell death spontaneously or induced by type 1 diabetes in FGF21 knockout (FGF21-KO) mice. Deletion of Fgf21 gene does not affect testicular cell proliferation, but significantly increases the spontaneous incidence of testicular TUNEL positive cells with increases in the Bax/Bcl2 expression ratio and apoptosis-inducing factor (AIF) expression. Diabetes induced significant increases in testicular TUNEL positive cells, Bax/Bcl2 expression ratio, AIF expression, CHOP and cleaved caspase-12 expression, and oxidative damage, but did not change the expression of cleaved caspase-3 and caspase-8. Deletion of Fgf21 gene also significantly enhances diabetes-induced TUNEL positive cells along with the increased expression of Bax/Bcl2 ratio, AIF, CHOP, cleaved caspase-12, and oxidative damage, which was significantly prevented by the supplementation of exogenous FGF21. These results suggest that Fgf21 gene may involve in maintaining normal spermatogenesis and also protect the germ cells from diabetes-induced apoptotic cell death probably via the prevention of diabetes-induced oxidative damage.     

Bis(4,7-dimethyl and 5-dinitro-1,10-phenanthroline) sulfato-oxovanadium(IV)-mediated in vivo male germ cell apoptosis

Oxovanadium(IV) [VO] complexes of 1,10-phenanthroline are a new class of potent apoptosis-inducing cytotoxic agents against human testicular cancer cells in vitro. The present study investigated the in vivo ability of four(bis)-chelated 1,10-phenanthroline [phen] complexes of sulfato-oxovanadium(IV)-VO(phen)(2), VO(Cl-phen)(2), VO(Me(2)-phen)(2) and VO(NO(2)-phen)(2)-with and without substitutions, to induce testicular germ cell apoptosis. Male germ cell loss in mice was measured by determining the epididymal sperm count, testicular weight and histological evaluation of the testes. Repetitive intratesticular injection (7.5 mg kg(-1) testis(-1)) of bis-chelated 1,10-phenanthroline complexes of oxovanadium(IV) with 4,7-dimethyl [VO(Me(2)-phen)(2)] and 5-dinitro [VO(NO(2)-phen)(2)] substitution led to decreased sperm counts and reduced testicular weights. Histopathological examination of testicular sections from VO(Me(2)-phen)(2)- and VO(NO(2)-phen)(2)-treated mice revealed a marked inhibition of spermatogenesis and preferential loss of maturing, as well as elongated spermatids. In situ evaluation of seminiferous tubule cross-sections by terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick end-labeling (TUNEL) and laser scanning confocal microscopy showed characteristic apoptotic germ cells delineating the periphery of the seminiferous tubules. The ability of bis-chelated 4,7-dimethyl- and 5-dinitro-substituted 1,10-phenanthroline complexes of oxovanadium(IV) to induce germ cell apoptosis in vivo may have potential utility in the treatment of human testicular germ cell tumors.     

Ascorbic acid protects against cadmium-induced endoplasmic reticulum stress and germ cell apoptosis in testes

Cadmium (Cd) is a testicular toxicant which induces endoplasmic reticulum (ER) stress and germ cell apoptosis in testes. This study investigated the effects of ascorbic acid on Cd-evoked ER stress and germ cell apoptosis in testes. Male mice were intraperitoneally injected with CdCl(2) (2.0mg/kg). As expected, a single dose of Cd induced testicular germ cell apoptosis. Interestingly, Cd-triggered testicular germ cell apoptosis was almost completely inhibited in mice treated with ascorbic acid. Interestingly, ascorbic acid significantly attenuated Cd-induced upregulation of GRP78 in testes. In addition, ascorbic acid significantly attenuated Cd-triggered testicular IRE1α and eIF2α phosphorylation and XBP-1 activation, indicating that this antioxidant counteracts Cd-induced unfolded protein response (UPR) in testes. Finally, ascorbic acid significantly attenuated Cd-evoked upregulation of CHOP and JNK phosphorylation, two components in ER stress-mediated apoptotic pathway. In conclusion, ascorbic acid protects mice from Cd-triggered germ cell apoptosis via inhibiting ER stress and UPR in testes.     

Involvement of germ cell apoptosis in the induction of testicular toxicity following hydroxyurea treatment

The present study investigated the occurrence of apoptotic cell death in the mouse testis at various intervals following the administration of hydroxyurea (HU). The presence of apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and by DNA fragmentation assay using ligation-mediated polymerase chain reaction. Both the incidence of apoptotic cells and the level of DNA fragmentation in the testis increased depending on the HU dose, and they were most apparent at the highest dose (400 mg/kg). The incidence of apoptotic cells in the HU-treated group increased continuously and peaked at 12 h, but then decreased gradually, reaching control levels by 48 h. After HU treatment, TUNEL-positive apoptotic cells increased in the seminiferous epithelium of the tubules, and affected cells were found synchronously in the tubules of animals treated with HU. Spermatogonia and spermatocytes were found to be affected selectively. TUNEL-positive cells were found to be stage-specific and were primarily in stage IV-VI tubules. It has been shown that in vivo HU exposure induced testicular germ cell apoptosis dose dependently in a time- and stage-specific manner, and damaged cells appeared to be eliminated by phagocytosis by neighboring cells. Apoptosis of damaged testicular germ cells is apparently a common response to various testicular toxicants therefore protecting the next generations of germ cells from the damaged cell population.     

Fas- or FasL-deficient mice display an increased sensitivity to nitrobenzene-induced testicular germ cell apoptosis

We have previously reported that the Fas/Apo-1/CD95-mediated apoptosis-inducing signaling system participates in the initiation of toxicant-induced testicular germ cell apoptosis. The contribution of Fas-mediated signaling is especially evident in the initiation of germ cell apoptosis after mono-(2-ethylhexyl)phthalate (MEHP)-induced Sertoli cell injury. In previous work, we demonstrated that the incidence of germ cell apoptosis after MEHP exposure is significantly reduced in B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of the FasL protein (the associated ligand that activates Fas). This has led to the hypothesis that activation of the Fas-mediated signaling pathway is a common mechanism for the initiation of germ cell apoptosis after toxicant-induced Sertoli cell injury. To test this hypothesis, we evaluated the sensitivity of testicular germ cells of wild-type, gld- and Fas-deficient CBA/KlJms-Tnfrsf6lpr-cg((lpr-cg)) (lpr(cg)) mice to undergo apoptosis after exposure to the Sertoli cell toxicant nitrobenzene (NB). Adult, 8-week-old gld mice treated with a single oral dose of NB (800 mg/kg) were observed to have a higher apoptotic index (AI; 66.1+/-1.3) 24 h after exposure as compared with the wild-type C57BL/6 (C57) mice (50.4+/-1.8). Similarly, 8-week-old lpr(cg) mice treated with NB displayed a higher AI 24 h after exposure (45.1+/-4.6) as compared with the wild-type CBA/KlJms (CBA) mice (32.1+/-3.8). Interestingly, exposure of both peri-pubertal 4-week-old C57 and gld mice showed a similar increase in the incidence of germ cell apoptosis after NB (600 mg/kg) exposure. Taken together, these findings indicate that Fas-mediated signaling is not required for NB-induced germ cell apoptosis and imply that a dysfunctional Fas signaling system sensitizes adult mice to NB-induced germ cell elimination.     

Apoptosis in male germ cells in response to cyclin A1-deficiency and cell cycle arrest

Male mice homozygous for a mutated allele of the cyclin A1 gene (Ccna1) are sterile due to a block in cell cycle progression before the first meiotic division. Meiosis arrest in Ccna1(-/-) spermatocytes is associated with desynapsis abnormalities, lowered MPF activity, and apoptosis as evidenced by TUNEL-positive staining. With time, adult testicular tubules exhibit severe degeneration: some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis. The mechanisms by which the cells sense the cell cycle arrest and the regulation of the decision to undergo cell death are under investigation.     

Lead-induced cell death in testes of young rats

Lead is a well-documented testicular toxicant. The present work was planned to study the occurrence of germ cell death after lead administration. Young growing rats were treated with 5, 10 and 20 mg kg(-1) body weight of lead for 2 weeks. Cell death was assessed by employing in situ TUNEL staining, DNA electrophoresis and morphological examination of the tubules. The results showed that Pb induced significant numbers of germ cells to undergo apoptosis in the seminiferous tubules of rats treated with 20 mg kg(-1) body weight. However, DNA fragmentation was not detected at any of the doses. The level of lead accumulation in the testis increased in a dose-dependent manner.     

Chemosensitization by fibroblast growth factor-2 is not dependent upon proliferation,S-phase accumulation,or p53 status

Fibroblast growth factor-2 (bFGF/FGF-2) is a pleiotropic growth factor that functions as a survival factor and directs apoptosis during embryogenesis and development. As a survival factor, FGF-2 would be expected to protect cells against drug toxicities. Such protection has been reported in some cells treated with some chemotherapeutic drugs. However, we recently demonstrated that FGF-2 can sensitize NIH 3T3 mouse fibroblasts to the cytotoxic and apoptotic effects of cisplatin. Sensitization requires prolonged incubation of cells with FGF-2 before the addition of cisplatin, and it requires an FGF-2 concentration (5-10 ng/mL) that is higher than that needed for its mitogenic effects (0.5 ng/mL). We now report that FGF-2 can also sensitize MCF7 human breast cancer cells and A2780 human ovarian cancer cells, as well as NIH 3T3 cells, to cisplatin. FGF-2 did not affect the cisplatin sensitivity of SKOV3 ovarian cancer cells or a panel of seven pancreatic cancer cell lines. We have demonstrated that the sensitizing effect is not simply a function of the mitogenic activity of FGF-2 on cells, as we did not observe sensitization with other growth-stimulatory factors (FGF-1 and epidermal growth factor); the sensitizing effect of FGF-2 was observed even with cell lines that were not growth-stimulated by FGF-2; and sensitization was not restricted to cells in S-phase of the cell cycle. These results indicate that cell proliferation is neither necessary nor sufficient for sensitization by FGF-2. Moreover, sensitization to cisplatin appears to be p53-independent, as p53-null 3T3 10-1 cells were equally sensitized by FGF-2. Finally, FGF-2 also sensitized NIH 3T3 and MCF7 cells to carboplatin, and had smaller effects on the sensitivity of these cell lines to doxorubicin and docetaxel. FGF-2 had no effect on sensitivity to etoposide in any cell line tested. Therefore, sensitization by FGF-2 was most effective with the platinum compounds, suggesting that this activity may be specific to particular mechanisms of drug action.     

Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII–VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway.     

Death receptor response in rodent testis after mono-(2-ethylhexyl) phthalate exposure

Apoptosis of testicular germ cells is critical for the maintenance of functional spermatogenesis. Previously, we have demonstrated that the Fas (Apo-1, CD95) receptor participates in the regulation of germ cell apoptosis, particularly after toxicant-induced Sertoli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague-Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague-Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFkappaB activation) revealed an early increase in NFkappaB-DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis (gld and lpr(cg)) reiterates the importance of the Fas signaling pathway.     

Cytotoxic effects of benzene on mouse germ cells determined by flow cytometry

Flow cytometric (FCM) DNA content measurements were performed on testicular monocellular suspensions obtained from mice exposed per os to 0, 1, 2, 4, 6, and 7 ml/kg body weight of benzene in order to investigate its cytotoxic action on germ cells. The effects of benzene were measured 7, 14, 21, 28, and 70 d after treatment. Benzene had no effect on testis weight, but FCM analysis showed the relative percentages of some cell subpopulations (tetraploid and haploid cells) to be different from the control pattern, indicating the occurrence of some cytotoxic damage to differentiating spermatogonia. These data demonstrate that spermatogenesis is sensitive to benzene single exposures as evidenced by an altered cell ratio of testicular cell types.     

Crosstalk between endoplasmic reticulum stress and mitochondrial pathway mediates cadmium-induced germ cell apoptosis in testes

Cadmium (Cd) is associated with male infertility and poor semen quality in humans. Increasing evidence demonstrates that Cd induces testicular germ cell apoptosis in rodent animals. However, the molecular mechanisms of Cd-induced testicular germ cell apoptosis remain poorly understood. In the present study, we investigated the role of endoplasmic reticulum (ER) stress on Cd-evoked germ cell apoptosis in testes. We show that spliced form of XBP-1, the target of the IRE1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an ER chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly caused eIF2α and JNK phosphorylation in testes, indicating that the unfolded protein response pathway in testes was activated by Cd. Interestingly, phenylbutyric acid (PBA), an ER chemical chaperone, attenuated Cd-induced ER stress and protected against germ cell apoptosis in testes. In addition, PBA significantly attenuated Cd-evoked release of cytochrome c from mitochondria to cytoplasm in testes. Taken together, these results suggest that crosstalk between ER stress signaling and mitochondrial pathway mediates Cd-induced testicular germ cell apoptosis.     

Mechanism of testicular protection of carvedilol in streptozotocin-induced diabetic rats

Aims:

Male sub-fertility and infertility are major complications of diabetes mellitus. The non-selective β-blocker carvedilol has been reported to have favorable effects on some of the diabetic complications based on its antioxidant and anti-apoptotic effects. This study aims to evaluate the possible testicular protective effect of carvedilol in streptozotocin (STZ)-induced diabetic rat model and its possible mechanisms.

Materials and Methods:

Diabetes was induced by a single i.p. dose of 65 mg/kg of STZ. In parallel groups of diabetic rats, carvedilol in low and high doses (1 and 10 mg/kg/day orally) were administered for 4 weeks. Oxidative stress markers as reduced glutathione (GSH) and the product of lipid peroxidation; malondialdehyde (MDA) were evaluated in testicular homogenate. The level of expression of the apoptotic marker; caspase 3, was assessed using western blot, followed by densitometric analysis.

Results:

Induction of diabetes caused distortion of histological normal testicular structure, with decrease (P < 0.05) in GSH and increase (P < 0.05) in MDA, as well as induction of caspase 3 expression. Carvedilol in low or high doses reverted diabetes-induced histological damage, restored antioxidant activity and ameliorated caspase 3 expression.

Conclusion:

Carvedilol confers testicular protection against diabetes-induced damage through antioxidant and anti-apoptotic mechanisms.KEY WORDS: Apoptosis, carvedilol, diabetes, oxidative stress, testes     

Inactivation of 14-3-3 protein exacerbates cardiac hypertrophy and fibrosis through enhanced expression of protein kinase C beta 2 in experimental diabetes

Diabetic cardiomyopathy is associated with cardiac hypertrophy and fibrosis. Activation of protein kinase C (PKC) has been implicated in the diabetes-induced cardiovascular complications. PKCbeta2 isoform is preferentially found to be activated in the diabetic myocardium. However, the role of PKCbeta2 in diabetic cardiomyopathy is not clear. 14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic and checkpoint control pathways, and have been shown to bind with PKC isozymes and negatively regulate their enzymatic activities. The present study tests whether 14-3-3 protein regulates cardiac hypertrophy and fibrosis in streptozotocin (STZ)-induced diabetic mice, using transgenic mice with cardiac specific over-expression of dominant negative (DN) 14-3-3 protein. In addition, we examined the relationship between 14-3-3 protein and PKCbeta2 in the diabetic myocardium. Cardiac myocyte diameter, content of cardiac fibrosis, left ventricular tissue expressions of atrial natriuretic peptide, transforming growth factor beta1, collagen III and PKCbeta2 were significantly elevated 28 and 56 d after STZ injection in transgenic DN-14-3-3 mice, when compared to their non-transgenic counterparts. These results clearly demonstrate that the functional inactivation of 14-3-3 protein in DN-14-3-3 mice exacerbates diabetes-induced cardiac hypertrophy and fibrosis. The exacerbations of cardiac hypertrophy and fibrosis were significantly and positively correlated with the enhanced expression of PKCbeta2 in DN-14-3-3 mice. Our results indicate for the first time that 14-3-3 protein negatively regulates cardiac hypertrophy and fibrosis, possibly through controlling the expression of PKCbeta2 in the diabetic myocardium.     

Studies on the characteristics and mechanisms of testicular toxicity induced by Hydroxyurea

Abstract

Objective: Apoptosis plays a dominant role in both spontaneous spermatogenesis and germ cell death. This study was aimed to investigate the functions of related genes in testicular germ cell death induced by Hydroxyurea (HU).

Method: Wild-type (WT) and FasL transgenic (TG) DBA/C57BL mice were intraperitoneal injected with 400?mg/kg HU. Twelve hours later, testes were collected. Histomorphology of testis was observed by staining with Periodic Acid Schiff (PAS). Apoptosis was assessed by TUNEL assay. mRNA and protein levels of related genes were evaluated by quantitative RT-PCR and Western blot, respectively.

Results: The 2?×?2 factorial design comparative experiments between the WT and TG mice showed that the TG mice exhibited a higher basal apoptotic index. The basal mRNA levels of Fas and FasL and protein levels of Fas, FasL, Caspase-3, Caspase-8 and Caspase-9 in the TG mice were also higher than that in the WT mice. Twelve hours after injection of HU, the testicular tubules exhibited no significantly morphological changes but apoptosis index remarkably increased in both the WT and TG mice, with the latter having the higher amplitude. Although, HU up-regulated the mRNA of apoptosis-related genes, such as Fas and FasL, in both the TG and WT mice, the increased amplitude was more obvious in the TG mice. By Western blot analysis, apoptosis-related proteins Fas, FasL Caspase-3, Caspase-8 and Caspase-9 were significantly increased in both the WT and TG mice, with the TG mice exhibiting a greater up-regulation.

Conclusion: Germ cell apoptosis induced by the HU treatment may be related to the FasL-mediated signal transduction pathway.     

N-乙酰半胱氨酸对急性乙醇暴露致小鼠睾丸损伤的保护作用

目的在成功建立乙醇诱导小鼠睾丸损伤模型的基础上,该研究主要探讨N-乙酰半胱氨酸(NAC)对急性乙醇暴露致小鼠睾丸损伤的保护作用。方法该研究由2个实验组成。实验1:28只雄性小鼠被随机分成4组,对照组和乙醇(1、3、6 g·kg-1)处理组;实验2:24只雄性小鼠被随机分成4组,对照组、乙醇组、NAC组和NAC+乙醇组。2个实验均于乙醇处理后24h剖杀小鼠,取睾丸称重,各组小鼠睾丸用MDF液固定,制作石蜡切片,以备后续睾丸HE染色和免疫组织化学检测。结果 3和6 g·kg-1乙醇暴露明显引起小鼠睾丸内多核巨细胞并向管腔内生殖细胞脱落等组织病理学改变;1和3 g·kg-1乙醇暴露明显抑制小鼠睾丸生殖细胞增殖;NAC明显保护3 g·kg-1乙醇暴露所致小鼠睾丸病理学损伤,显著减轻3 g·kg-1乙醇暴露对小鼠睾丸细胞增殖的抑制作用。结论 NAC明显保护乙醇急性暴露对小鼠睾丸组织病理学损伤。