Sirt1 deficiency attenuates spermatogenesis and germ cell function

In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (-/-) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced ( approximately 2-10-fold less; P相似文献   

Transient protection from heat-stress induced apoptotic stimulation by metastasis-associated protein 1 in pachytene spermatocytes

Background

Deregulated thermal factors have been frequently implicated in the pathogenesis of male infertility, but the molecular basis through which certain responses are directed remain largely unknown. We previously reported that overexpression of exogenous Metastasis-associated protein 1 (MTA1) protects spermatogenic tumor cells GC-2spd (ts) against heat-induced apoptosis. To further dissect the underlying mechanism, we addressed here the fine coordination between MTA1 and p53 in pachytene spermatocytes upon hyperthermal stimulation.

Methodology/Principal Findings

High level of MTA1 expression sustained for 1.5 h in primary spermatocytes after heat stress before a notable decrease was detected conversely correlated to the gradual increase of acetylation status of p53 and of p21 level. Knockdown of the endogenous MTA1 in GC-2spd (ts) elevated the acetylation of p53 by diminishing the recruitment of HDAC2 and thereafter led to a dramatic increase of apoptosis after heat treatment. Consistent with this, in vivo interference of MTA1 expression in the testes of C57BL/6 mice also urged an impairment of the differentiation of spermatocytes and a disruption of Sertoli cell function due to the elevated apoptotic rate after heat stress. Finally, attenuated expression of MTA1 of pachytene spermatocytes was observed in arrested testes (at the round spermatid level) of human varicocele patients.

Conclusions

These data underscore a transient protective effect of this histone modifier in primary spermatocytes against heat-stress, which may operate as a negative coregulator of p53 in maintenance of apoptotic balance during early phase after hyperthermal stress.     

Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction

Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment.     

Interplay between p53 and Ink4c in spermatogenesis and fertility

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.     

MST1 promotes apoptosis through regulating Sirt1-dependent p53 deacetylation

Mammalian Sterile 20-like kinase 1 (MST1) protein kinase plays an important role in the apoptosis induced by a variety of stresses. The MST1 is a serine/threonine kinase that is activated upon apoptotic stimulation, which in turn activates its downstream targets, JNK/p38, histone H2B and FOXO. It has been reported that overexpression of MST1 initiates apoptosis by activating p53. However, the molecular mechanisms underlying MST1-p53 signaling during apoptosis are unclear. Here, we report that MST1 promotes genotoxic agent-induced apoptosis in a p53-dependent manner. We found that MST1 increases p53 acetylation and transactivation by inhibiting the deacetylation of Sirtuin 1 (Sirt1) and its interaction with p53 and that Sirt1 can be phosphorylated by MST1 leading to the inhibition of Sirt1 activity. Collectively, these findings define a novel regulatory mechanism involving the phosphorylation of Sirt1 by MST1 kinase which leads to p53 activation, with implications for our understanding of signaling mechanisms during DNA damage-induced apoptosis.     

Quinestrol induces spermatogenic apoptosis in vivo via increasing pro-apoptotic proteins in adult male mice

The effects of quinestrol on spermatogenesis were investigated in adult male mice by daily intragastric administration of quinestrol with various doses of 5, 10, 50 and 100 mg/kg body weight for 10 days. The sperm counts declined while the number of abnormal spermatozoa went up in mice treated with quinestrol. The testicular weight and seminiferous tubular area gradually declined with increasing dosages of quinestrol to 50 and 100 mg/kg. Rarefied germ cells showed irregular distributions in the seminiferous tubules of mice treated with 50 and 100 mg/kg quinestrol. Apoptosis was highly pronounced in spermatogonia, spermatocytes, spermatids and Leydig cells. Antioxidant enzyme activities – superoxide dismutase and glutathione peroxidase – as well as total antioxidant capacity significantly reduced, while malondialdehyde contents increased. The number of germ cells expressing caspase-3, p53, Bax and FasL significantly increased whereas cells expressing Bcl-2 significantly decreased in groups treated with 50 and 100 mg/kg quinestrol compared with the control. The concentration of nitrogen monoxidum also increased significantly under these dosages. The results suggest that quinestrol stimulates oxidative stress to induce apoptosis in spermatogenic cells through the mitochondrial and death receptor pathways in adult male mice.     

Panorama de l’infertilité masculine

This paper reviews new epidemiological, etiological and therapeutic aspects of male infertility. Because of the great improvement in the efficacy of assisted reproductive techology due to ICSI, the recently discovered genetic causes of male infertility have to be considered. While studies concerning the role of xenobiotics in disrupting endocrine regulation of testicular functions are in progress, genetic causes of male infertility has been discovered. Microdeletions of the long arm of the Y chromosome account for a substantial part of unexplained spermatogenic failures. Mutations of CFTR gene are involved in bilateral agenesis of vas deferens. This condition might be considered as a mild form of cystic fibrosis. These genetic defects together with chromosmal abnormalities, which are known to be responsible for spermatogenic failures, should be considered as potential sources of reproductive abnormalities of more global pathology transmissible to children who can be obtained by ICSI with ejaculated, epididymal or testicular sperm.     

Limited role of Sirt1 in cancer protection by dietary restriction

Dietary restriction (DR) has multiple beneficial effects, the two most prominently studied being an increased longevity and an increased cancer protection. Mammalian Sirt1 is a protein deacetylase that has been linked to DR. To explore the relation between Sirt1 and DR, we have examined here DR-induced cancer protection in mice overexpressing Sirt1 (2-3 fold) under its own regulatory elements (Sirt1-tg mice). In particular, we have subjected p53?deficient mice, carrying or not the Sirt1-tg allele, to every-other-day fasting (EOD), which is a type of DR that significantly delays cancer onset. As expected, EOD extended the survival of p53-heterozygous (p53^+/-) mice. However, the extension of survival of p53-heterozygous mice by EOD was the same in the presence or absence of the Sirt1-tg allele. These results suggest that Sirt1 has a limited role in mediating cancer protection by DR in mammals.     

Epimedium protects against dyszoospermia in mice with Pex3 knockout by exerting antioxidant effects and regulating the expression level of P16

Oxidative stress (OS) is one of the primary factors leading to male infertility. Oral administration of antioxidants has thus far been found to significantly improve the quality of human sperm. Therefore, antioxidant treatment has become the consensus among international experts on male infertility. In this study, peroxisomal biogenesis factor 3 (Pex3)-knockout (KO, −/−) mice were used as a model to compare the efficacy of three types of traditional Chinese medicine (TCM) granules (Epimedium [YYH], Cuscuta [TSZ], and Rhodiola [HJT]) for male reproductive function rescue. YYH was revealed to be the best and exerted a rescue effect on Pex3−/− mice with spermatogenesis defects. In addition, YYH prominently reduced ROS levels in the testes, inhibited DNA oxidative damage in spermatogenic cells, promoted the proliferation of spermatogenic cells, and inhibited apoptosis in Pex3−/− male mice. Furthermore, the mechanism by which YYH ameliorated dyszoospermia was confirmed via the establishment of cyclin-dependent kinase inhibitor 2 A (P16Ink4a)-KO mice. Specifically, Pex3−/− mice produced elevated amounts of ROS, which damaged germ cell DNA and further activated the signaling pathway of the cell senescence regulatory protein P16-CDK6, resulting in cell cycle arrest and eventually contributing to spermatogenesis dysfunction. YYH supplementation partially corrected the associated phenotype in gene KO mice by affecting P16 expression levels, thus improving the reproductive outcome to a certain extent.Subject terms: Senescence, Embryology, Infertility, Experimental models of disease, Translational research     

Resveratrol inhibits ferroptosis and decelerates heart failure progression via Sirt1/p53 pathway activation

Resveratrol is an organic compound widely studied for its therapeutic uses. We investigated whether resveratrol exerts cardioprotective effects by inhibiting ferroptosis via the Sirt1/p53 pathway. A heart failure model was established by aortic coarctation in Sirt1 knockout mice. The superoxide dismutase (SOD), glutathione (GSH) levels and mitochondrial morphology in murine heart tissues were assessed at different time points to determine the role of ferroptosis in heart failure progression. The cardiac function of mice with heart failure was evaluated by determining the brain natriuretic peptide (BNP) and sST2 concentration and conducting echocardiography. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transfected with the p53 K382R mutant and Sirt1 interference lentiviral vectors. Immunoprecipitation (IP) experiments were performed to investigate whether Sirt1 influences ferroptosis via p53 K382 acetylation and SLC7A11 expression modulation. Resveratrol improved cardiac function in mice and decelerated ferroptosis and fibrosis progression in heart failure. However, the ability of resveratrol to prevent ferroptosis and treat heart failure was lost after silencing Sirt1. Sirt1 reduced ferroptosis by diminishing the levels of p53 K382 acetylation, reducing the degradation of SLC7A11, and increasing the levels of GSH and glutathione peroxidase 4 (GPX4) in cells. In conclusion, by activating the Sirt1/p53 pathway in heart failure, resveratrol decreased the depletion of SLC7A11, inhibited ferroptosis, and improved cardiac function.     

The expression of PAC1 increases in the degenerative thymus and low dose PACAP protects female mice from cyclophosphamide induced thymus atrophy

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with cytoprotective ability mediated by its specific receptor PAC1. In this research, firstly the thymus index and the expression of PAC1 in the normal and degenerative thymus with different gender were assayed; secondly PACAP in different dose was used to treat the female mice with cyclophosphamide (CPS) and the changes in thymus index, the expression of PAC1, histopathology, apoptosis, oxidative status and the caspase 3 activity in thymus were determined and compared. It was found that in the mice of age from 1 to 9 weeks in the stage of sex development, the thymus index was significantly higher in female mice than in male mice. And it was found for the first time that the PAC1 expression level in thymus of female mice was significantly higher than that of male mice and the expression of the PAC1 and PACAP increased significantly in the degenerative thymus induced by CPS. After PACAP was co-injected with CPS to the female mice, it was shown that only low dose (1 nmol/kg) of PACAP promoted the thymus index, inhibited the cell apoptosis, ameliorated the oxidative status and decreased the caspase activity significantly, while high dose (10 nmol/kg) of PACAP had no significant protective effects against CPS-induced thymus atrophy. It was concluded that the expression of PAC1 in the thymus changes in reverse ratio with thymus index and in direct ratio with cell apoptosis and only low dose of PACAP had positive effects against the CPS-induced thymus atrophy.     

Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 μg/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32°C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL. This work was supported, in part, by grants from the National Institute of Environmental Health Sciences/NIH (ES09145, JHR), Toxicology Training grant (ES T32 ES007247, CM), NIH Center Grant (P30 ES07784^, JHR) and the Center for Molecular and Cellular Toxicology (CMCT).     

Microinjection manipulation resulted in the increased apoptosis of spermatocytes in testes from intracytoplasmic sperm injection (ICSI) derived mice

The invention of intracytoplasmic sperm injection (ICSI) has possibly been the most important development in reproductive medicine, one that has given hope to thousands of infertile couples worldwide. However, concerns remain regarding the safety of this method since it is a more invasive procedure than in vitro fertilization (IVF), since a spermatozoon is injected into the oocyte cytoplasm. Using mice derived from IVF technology as a control, we assessed the influence of invasive microinjection in the process of transferring sperm into oocyte cytoplasm in ICSI procedure on the development and physiologic function of resultant offspring. Our results demonstrated that mice produced from ICSI and IVF had no significant difference in phenotypic indices including body weight, forelimb physiology, and learning and memory ability. However, increased spermatocyte apoptosis was observed in the testis of adult ICSI mice, when compared with IVF mice. And, decreased testis weight and marked damage of spermatogenic epithelia were found in aged ICSI mice. Furthermore, proteomic analysis verified that most of the differentiated proteins in testes between adult ICSI and IVF mice were those involved in regulation of apoptosis pathways. Our results demonstrated that the microinjection manipulation used in the ICSI procedure might pose potential risks to the fertility of male offspring. The changed expression of a series of proteins relating to apoptosis or proliferation might contribute to it. Further studies are necessary to better understand all the risks of ICSI.     

Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling

Approximately half of all infertility cases can be attributed to male reproductive dysfunction for which low sperm count is a major contributing factor. The current study identified receptor-mediated lysophosphatidic acid (LPA) signaling as a new molecular component influencing male fertility. LPA is a small signaling phospholipid, the effects of which are mediated through at least five G protein-coupled receptors, named LPA 1-5. LPA1/2/3, but not LPA4/5, show high expression in mouse testis. Mice deficient in LPA1/2/3 showed a testosterone-independent reduction of mating activity and sperm production, with an increased prevalence of azoospermia in aging animals. A significant increase of germ cell apoptosis also was observed in testes. Germ cell apoptosis led to a reduction in germ cell proliferation. These data demonstrate a novel in vivo function for LPA signaling as a germ cell survival factor during spermatogenesis.     

宁夏密点麻蜥不同部位含药大鼠血清对人胃癌细胞凋亡的影响研究

为探讨宁夏密点麻蜥不同部位含药大鼠血清对人胃癌细胞SGC-7901凋亡影响及其抗癌机制。研究选取SPF级雄性SD大鼠分别以生理盐水、宁夏密点麻蜥不同部位水煎液灌胃,制备含药血清加于胃癌细胞,通过MTT检测细胞活性,AnnexinV-FITC/PI双染法检测细胞凋亡,Western-blot检测胃癌细胞Sirt1和P53蛋白表达。结果显示,宁夏密点麻蜥不同部位各组可明显降低Sirt1和P53蛋白表达水平,抑制细胞增殖,促进凋亡,以尾部组最明显。综上所述,宁夏密点麻蜥不同部位可能通过抑制SIRT1,降低P53,诱导细胞凋亡,其中以宁夏密点麻蜥尾部抗肿瘤作用最显著。