DNA甲基化在精子发生中的作用

精子发生(spermatogenesis)是一个高度特化的细胞复杂分化过程,其中DNA二核苷酸CpG甲基化变化与基因转录激活、染色质改构以及遗传印记相关,并且该甲基化与基因表达之间的关系是非直接的,其可通过染色质结构的改变或DNA与蛋白质的相互作用来介导。本文着重介绍精子发生过程中DNA甲基化及其跨代遗传风险、DNA甲基转移酶的调控机制以及DNA甲基化与男性不育之间的关系等,为不育症的防治、精子表观遗传质量评价以及降低辅助生殖技术后代表观遗传疾病风险等提供基础资料。     

DNA methylation changes in genes coding for leptin and insulin receptors during metabolic-altered pregnancies

The overwhelming rates of obesity worldwide are a major concern due to the elevated medical costs associated and the poor quality of life of obese patients. In the recent years, it has become evident that the intrauterine milieu can have a long-term impact on the foetus health. The placenta is a highly dynamic organ; whose primary function is to carry nutrients from the mother to the foetus and to remove waste products from the foetus. Any alteration in maternal circulating metabolites elicits a response in order to ensure the developing foetus an adequate growth environment. This response can be translated into epigenetic modifications in coding genes for metabolic-related receptors located in the placenta and foetal tissues. The most studied receptors involved in the metabolic sensing are the leptin and the insulin receptors. A maternal metabolic disease-like state can alter the expression of these receptors in different organs, including placenta. There is evidence that these alterations not only affect the expression level of these receptors, but there are also differences in epigenetic marks in regulatory elements of these genes that may become permanent despite the mother's treatment. This review provides evidence about possible mechanisms involved in the foetal programming of metabolic diseases originated from the pre-natal environment that could contributive to increasing levels of obesity in the world.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Circadian expression of DNA methylation and demethylation genes in zebrafish gonads

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01~ZT22:17 h), except for dnmt7 in males (ZT2:25 h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26~ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.     

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.     

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.     

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression.

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.     

Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis

Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here revealed that different combinations of histone variant genes are expressed in distinct spermatogenic cell types accompanying the progression of self-renewal and differentiation of SSCs, suggesting a systematic regulatory role histone variants play during spermatogenesis.     

Developmental regulation of expression of the lactate dehydrogenase (LDH) multigene family during mouse spermatogenesis

Expression of the Lactate Dehydrogenase (LDH) genes during various stages of spermatogenesis was studied by using a combination of Northern blot analyses and in situ hybridization techniques. These studies have indicated that developmentally programmed expression of all three functional LDH genes occurs during differentiation of germ cells. The LDH/C (ldh-3) gene was expressed exclusively during meiosis and spermiogenesis, beginning in leptotene/zygotene spermatocytes and continuing through to the elongated spermatids. LDH/C (ldh-3) gene expression was accompanied by transient expression of the LDH/A (ldh-1) gene in pachytene spermatocytes and round spermatids. The LDH/B (ldh-2) gene was expressed mainly in Sertoli and spermatogonial cells. By using somatic cell hybrids, the LDH/C (ldh-3) gene has been mapped to mouse chromosome 7, establishing that it is syntenic with the LDH/A (ldh-1) gene locus. Experimental observations made in this study provide new insight into the order and sequence of events involved in the regulation of gene expression of the LDH gene family during spermatogenesis.     

Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.     

Expression of genes coding melatonin and serotonin receptors in rodent skin

Targeted search for expression of melatonin and serotonin receptors genes in the skin of C57BL/6J mice showed expression of MT1B (but not MT1A). Mouse skin and hamster melanomas also expressed 5HT2B and 5HT7. We identified two novel isoforms of MT1A and 5HT7.     

A comparison of potato and vertebrate lactate dehydrogenases.

The incorporation of labelled leucine was measured in protein fractions of muscle in intact control and dystrophic female hamsters and also in cell-free preparations obtained from these animals. The labelling of the soluble sarcoplasmic protein fraction, the microsomal protein fraction and the sarcolemma protein fraction was increased in the dystrophic hindleg muscle. The specific radioactivities of the sarcolemma protein fraction and other fractions were increased markedly relative to that of free leucine in the dystrophic muscle. In cell-free preparations where ribonuclease effects were avoided, the dystrophic muscle exhibited an increased synthesis of peptide bonds.