Inhibition of CDC2/Cyclin B1 in response to selenium-induced oxidative stress during spermatogenesis: potential role of Cdc25c and p21

Various cell cycle regulators control and coordinate the process of cell cycle. Because of the crucial involvement of CDC2, Cyclin B1, Cdc25c, and p21 in cell cycle regulation, the present study was aimed to investigate the possibility that selenium (Se)-induced oxidative stress mediated alterations in Cdc25c and p21 may cause modulations in the CDC2/Cyclin B1 complex responsible for G2/M phase checkpoint during meiosis I of spermatogenesis. To create different Se status-deficient, adequate and excess Se, male Balb/c mice were fed yeast based Se deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet feeding schedule, a significant decrease in the Se and glutathione peroxidase levels were observed in the Se deficient group (I), whereas Se excess group (III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation (LPO) were seen in both group I and group III when compared to group II, thus indicating oxidative stressed conditions. The mRNA and protein expression of CDC2, Cyclin B1, and Cdc25c were found to be significantly decreased in groups I and III. However, the expression of p21, a kinase inhibitor, was found to be elevated in Se deficient and Se excess fed groups. A statistically significant decrease in the CDC2 kinase activity was also seen in the Se deficient and excess groups. These findings suggest that under the influence of Se-induced oxidative stress, the down regulation of CDC2/Cyclin B1 complex is mediated through changes in Cdc25c and p21 leading to the cell cycle arrest and thus providing new dimensions to the molecular mechanisms underlying male infertility.     

Diminished reproductive potential of male mice in response to selenium‐induced oxidative stress: Involvement of HSP70, HSP70‐2, and MSJ‐1

The oxidative stress imposed by nutritional variations in selenium (Se) has plausible role in reproductive toxicology and affects the reproductive potential. Also, the expression of heat shock proteins (HSPs) is a highly regulated event throughout the process of spermatogenesis and is modulated by stressful stimuli. This prompted us to investigate the possibility that Se‐induced oxidative stress may affect the fertility status by altering the expressions of the constitutive and inducible HSP70 proteins, having crucial role in spermatogenesis. Different Se status‐deficient, adequate, and excess, male Balb/c mice were created by feeding yeast‐based Se‐deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet‐feeding schedule, a significant decrease in the Se and glutathione peroxidase (GSH‐Px) levels was observed in the Se‐deficient group (I), whereas Se‐excess group (III) demonstrated an increase. Increased levels of reactive oxygen species, malondialdehyde, and alterations in the redox status in both groups I and III indicated oxidative‐stressed conditions. There was an overall reduced fertility status in mice supplemented with Se‐deficient and Se‐excess diet. The mRNA and protein expression of HSP70 was found to be elevated in these two groups, whereas the expression patterns of HSP70‐2 and MSJ‐1 demonstrated a reverse trend. In vitro CDC2 kinase assay showed reduced kinase activity in group I and group III. These findings suggest that Se‐induced oxidative stress by differentially regulating various HSP70s can affect its downstream factors having crucially important role in differentiation of germ cells and completion of spermatogenesis. Therefore, it can provide an insight into the mechanism(s) by which the oxidative stress–induced reproductive toxicity can lead to increased apoptosis/growth arrest and infertility. This will thus add new dimensions to the molecular mechanism underlying the human male infertility and open new vistas in the development of various chemo‐preventive methods. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:125–136, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20276     

Effects of maternal and dietary selenium (Se-enriched yeast) on the expression of p34(cdc2) and CyclinB1 of germ cells of their offspring in goats

The aim of the study was to evaluate the effect of selenium on the expression of p34^cdc2 and CyclinB1 (two components of MPF regulating cell cycle) of germ cells of their offspring in goats. A herd of 119 Taihang Black Goats, which was randomly divided into 4 treatments, received experimental diet with different Se levels (from Se-enriched yeast) for 174 d. The four treatments, fed with a basal diet, were supplemented with 0(control), 0.5, 2 and 4 mg kg^?1 DM Se. Testis samples were collected from the young male goats of each treatment group at the end of the study (30 d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. Results show that a significant decrease was observed in mRNA expression of p34^cdc2 and CyclinB1 in the testis of Se-deficient (Group 1) and Se-excess (Group 4) animals compared with that in Groups 2 and 3. However, no significant changes were found in mRNA expression of p34^cdc2 between Se-deficient (Group 1) and Se-excess (Group 4). Also the immunohistochemistry assay detected similar results of protein expression of these two genes. These results suggest, that maternal and dietary Se-induced oxidative stress can modulate the mRNA and protein expression of the cell cycle related genes (p34^cdc2 and CyclinB1) in the testis of their offspring. In addition, Se deficiency and Se excess could prevent the completion of the cell cycle.     

Effect of oxidative stress on the spermatogenic process and hsp70 expressions in mice testes

The effect of oxidative stress on the process of spermatogenesis in terms of hsp70 expression was studied. For creating different oxidative stressed mice, three selenium (Se) levels viz., deficient (group I), adequate (group II) and excess (group III) were fed for 8 weeks in a yeast-based diet. After completion of diet feeding, Se level was significantly decreased in group I and significantly increased in group III, as compared to group II. Glutathione peroxidase (GSH-Px) activity was significantly decreased in both liver and testis in group I animals; however, the activity was comparable in groups II and III. Significant increase in the testis glutathione-S-transferase (GST) activity was observed in group I. No change was seen in group III, when compared to group II. Histological analysis of testis revealed a significant decrease in the germ cell population in group I, as compared to group II, with a predominant effect on spermatid and mature sperm numbers. In group III, displacement of germ cell population was observed. ELISA assays for hsp70 level showed increase in group I as compared to group II, whereas no significant change was observed in group III, as compared to group II. Immunohistochemical analysis revealed intense localization of hsp70 only in spermatid and sperm cells. The expression in groups II and III was homogeneous with slightly increased expression around lumen in group III. The data indicate that excessive oxidative stress in Se deficient group, affects the spermatogenesis process, especially affecting the mature sperm number which in turn leads to infertility.     

Effect of selenium-induced oxidative stress on the oxidation reduction system and reproductive ability of male mice

The present study was carried out to evaluate the effect of selenium (Se)-induced oxidative stress on the oxidation reduction system and the fertility status of male mice. Different levels of Se, a potent antioxidant, were fed in three separate groups for 8 wk to create the different oxidative stress in mice. A significant decrese in the glutathione peroxidase (GSH-Px) in both liver and testis was observed in the Se-deficient (0.02 ppm) group I, whereas enzyme levels in the Se-excess (1 ppm) group were comparable to the Se-adequate (0.2 ppm) group. Glutathione-S-transferase activity was enhanced in group I in comparison to group II; however, no change was seen in group III. The glutathione reductase and superoxide dismutase activities were decreased in the Se-deficient group, whereas the enzyme levels were significantly increased in the Se-excess group. The fertility status of the animals studied in terms of percentage fertility and litter size showed a significant decrease in the reproductive ability of male mice in group I when compared to group II. No changes in the fertility status of animals were observed in group III. Thus, the data clearly indicate the effect of oxidative stress generated by feeding various Se levels on the oxidation reduction system and, consequently, its effect on the reproductive ability of male mice.     

Effect of experimental oxidative stress on steroidogenesis and DNA damage in mouse testis

The objective of the present study was to evaluate the effect of oxidative stress induced by feeding various levels of selenium on steroidogenesis and DNA damage in mouse testes. To create various levels of oxidative stress in mice, diets with three different Se levels were fed to separate groups for 8 weeks. Group 1 animals were fed a yeast-based diet, which was considered a Se-deficient diet (0.02 ppm). Group 2 and 3 animals were fed a Sedeficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively. After completion of the diet feeding, estimations were carried out, and results were compared with those of group 2. A significant decrease in Se levels was observed in group 1 animals, whereas they were greatly enhanced in group 3. Glutathione peroxidase (GSH-Px) activity was greatly reduced in both the liver and testes in group 1, whereas no significant changes were found in GSH-Px activity in group 3. Serum luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone levels were reduced in group 1. Significant decreases of sperm number and motility were observed in group 1 when compared to group 2 male mice. No changes in these parameters were observed in group 3. DNA fragmentation was observed in both groups 1 and 3; however, the damage was more prevalent in group 1. The results clearly demonstrate the effect of oxidative stress generated by feeding various Se levels on the steroidogenesis and DNA fragmentation in mice testes.     

Spermatogenesis in goats with and without scrotum bipartition

The objective of the present research was to quantify the seminiferous epithelium cells, spermatogenesis efficiency and characterize the ultrastrucure of Sertoli cells in goats. Eighteen goats were used and divided into three groups: Group I - goats without bipartition of the scrotum; Group II - animals with bipartition of the scrotum in up to 50% of the testicular length; Group III - goats with bipartition of the scrotum in more than 50% of the testicular length. The goat testes in Group III had a greater number of primary spermatocytes (25.37 ± 4.55 cells per cross sections), spermatids (112 ± 15.12 cells per cross sections), and Sertoli cells (9.46 ± 1.74 cells per cross sections) than the animals in Groups I and II (P<0.05). The spermatogenic mitotic, meiotic, and general efficiency were greater in animals in Group III (1.25 ± 0.28; 5.12 ± 1.63; 6.44 ± 1.96) when compared to those in Groups I and II. Sheet-like processes originated from the Sertoli cell body as simple and smooth structures which involved almost all the surface of germ cells. Slender cord-like processes originated from Sertoli cells and also from the sheet-like processes. The relative frequency of the cycle stages showed differences among the groups of goats studied, and the highest frequency was in Stage 3 (20.68% for goats in Group I, 21.15% for those in Group II, and 16.89% for the animals in Group III). In conclusion, goats with bipartition of the scrotum have a greater number of germ and Sertoli cells per cross section of seminiferous tubule, that indicated a greater sperm production when compared to the other groups, and the ultrastructure of the Sertoli cell process did not present any relationship with bipartition of the scrotum.     

Ameliorative Effects of Selenium on Cadmium-Induced Injury in the Chicken Ovary: Mechanisms of Oxidative Stress and Endoplasmic Reticulum Stress in Cadmium-Induced Apoptosis

Despite the well-established toxicity of cadmium (Cd) to animals and the ameliorative effects of selenium (Se), some specific mechanisms in the chicken ovary are not yet clarified. To explore the mechanism by which the toxicity effect of Cd is induced and explore the effect of supranutritional Se on Cd toxicity in female bird reproduction, forty-eight 50-day-old Isa Brown female chickens were divided randomly into four groups. Group I (control group) was fed the basic diet containing 0.2 mg/kg Se. Group II (Se-treated group) was fed the basic diet supplemented with sodium selenite (Na₂SeO₃), and the total Se content was 2 mg/kg. Group III (Se + Cd-treated group) was fed the basic diet supplemented with Na₂SeO₃; the total Se content was 2 mg/kg, and it was supplemented with 150 mg/kg cadmium chloride (CdCl₂). Group IV (Cd-treated group) was with the basic diet supplemented with 150 mg/kg CdCl₂. The Cd, estradiol (E2), and progestogen (P4) contents changed after subchronic Cd exposure in chicken ovarian tissue; subsequently, oxidative stress occurred and activated the endoplasmic reticulum (ER) pathway to induce apoptosis. Further, Se decreased the accumulation of Cd in ovarian tissue, increased the E2 and P4 contents, alleviated oxidative stress, and reduced apoptosis via the ER stress pathway. The present results demonstrated that Cd could induce apoptosis via the ER stress pathway in chicken ovarian tissue and that Se had a significant antagonistic effect. These results are potentially valuable for finding a strategy to prevent Cd poisoning.     

Induction of apoptotic death by curcumin in human tongue squamous cell carcinoma SCC-4 cells is mediated through endoplasmic reticulum stress and mitochondria-dependent pathways

Curcumin from the rhizome of the Curcuma longa plant has been noted for its chemo-preventative and chemo-therapy activities, and it inhibits the growth of many types of human cancer cell lines. In this study, the mechanisms of cell death involved in curcumin-induced growth inhibition, including cell cycle arrest and induction of apoptosis in human tongue cancer SCC-4 cells, were investigated. Herein, we observed that curcumin inhibited cell growth of SCC-4 cells and induced cell death in a dose-dependent manner. Treatment of SCC-4 cells with curcumin caused a moderate and promoted the G(2) /M phase arrest, which was accompanied with decreases in cyclin B/CDK1 and CDC25C protein levels. Moreover, curcumin significantly induced apoptosis of SCC-4 cells with a decrease of the Bcl-2 level, reduction of mitochondrial membrane potential (ΔΨ(m) ), and promoted the active forms of caspase-3. Curcumin also promoted the releases of AIF and Endo G from the mitochondria in SCC-4 cells by using confocal laser microscope. Therefore, we suggest that curcumin induced apoptosis through a mitochondria-dependent pathway in SCC-4 cells. In addition, we also found that curcumin-induced apoptosis of SCC-4 cells was partly through endoplasmic reticulum stress. In conclusion, curcumin increased G(2) /M phase arrest and induced apoptosis through ER stress and mitochondria-dependent pathways in SCC-4 cells.     

Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats

The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson’s trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.     

内质网应激与哺乳动物雄性生殖

内质网应激 (endoplasmic reticulum stress,ERS) 激活未折叠蛋白反应,维持哺乳动物细胞的胞内稳态,过度持续的ERS导致细胞凋亡。最新研究表明,ERS对哺乳动物雄性生殖有重要的调节作用,包括对精母细胞、睾丸结构及精子发生的影响。ERS是研究生殖细胞生存和凋亡的新通路。雄性不育可能是由过度ERS引起的。本文通过简述ERS的最新研究进展,分析雄性生殖与ERS的关系,并从ERS调控雄性生殖角度提出新的理解和展望。     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Social isolation-induced aggression potentiates anxiety and depressive-like behavior in male mice subjected to unpredictable chronic mild stress

Background

Accumulating epidemiological evidence shows that life event stressors are major vulnerability factors for psychiatric diseases such as major depression. It is also well known that social isolation in male mice results in aggressive behavior. However, it is not known how social isolation-induced aggression affects anxiety and depressive-like behavior in isolated male mice subjected to unpredictable chronic mild stress (CMS), an animal model of depression.

Methodology/Principal Findings

C57/B6 male mice were divided into 3 groups; non-stressed controls, in Group I; isolated mice subjected to the CMS protocol in Group II and aggression by physical contact in socially isolated mice subjected to the CMS protocol in Group III. In the sucrose intake test, ingestion of a 1% sucrose solution by mice in Groups II and III was significantly lower than in Group I. Furthermore, intake of this solution in Group III mice was significantly lower than in Group II mice. In the open field test, mice in Group III, showed reduced locomotor activity and reduced entry and retention time in the central zone, compared to Groups I and II mice. Moreover, the distances moved in 1 hour by Group III mice did not differ between night and morning. In the light/black box test, Groups II and III animals spent significantly less time in the light box compared to Group I animals. In the tail suspension test (TST) and forced swimming test (FST), the immobility times of Group II and Group III mice were significantly longer than in Group I mice. In addition, immobility times in the FST were significantly longer in Group III than in Group II mice.

Conclusions/Significance

These findings show that social isolation-induced aggression could potentiate anxiety and depressive -like behaviors in isolated male mice subjected to CMS.     

Expression of Cell Cycle-Related Genes During Neuronal Apoptosis: Is there a Distinct Pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

Mammalian E-type Cyclins Control Chromosome Pairing,Telomere Stability and CDK2 Localization in Male Meiosis

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.