GRP78/IRE1 and cGAS/STING pathway crosstalk through CHOP facilitates iodoacetic acid-mediated testosterone decline.

Iodoacetic acid (IAA) is an emerging unregulated iodinated disinfection byproduct with high toxicity and widespread exposure. IAA has potential reproductive toxicity and could damage male reproduction. However, the underlying mechanisms and toxicological targets of IAA on male reproductive impairment are still unclear, and thus Sprague-Dawley rats and Leydig cells were used in this work to decode these pending concerns. Results showed that after IAA exposure, the histomorphology and ultrastructure of rat testes were abnormally changed, numbers of Leydig cells were reduced, the hypothalamic-pituitary-testis (HPT) axis was disordered, and testosterone biosynthesis was inhibited. Proteomics analyses displayed that oxidative stress, endoplasmic reticulum stress, and steroid hormone biosynthesis were involved in IAA-caused reproductive injury. Antioxidant enzymes were depleted, while levels of ROS, MDA, 8-OHdG, and γ-H2A.X were increased by IAA. IAA triggered oxidative stress and DNA damage, and then activated the GRP78/IRE1/XBP1s and cGAS/STING/NF-κB pathways in Leydig cells. The two signaling pathways constructed an interactive network by synergistically regulating the downstream transcription factor CHOP, which in turn directly bound to and negatively modulated steroidogenic StAR, finally refraining testosterone biosynthesis in Leydig cells. Collectively, IAA as a reproductive toxicant has anti-androgenic effects, and the GRP78/IRE1 and cGAS/STING pathway crosstalk through CHOP facilitates IAA-mediated testosterone decline.

The miRNA-29b Is Downregulated in Placenta During Gestational Diabetes Mellitus and May Alter Placenta Development by Regulating Trophoblast Migration and Invasion Through a HIF3A-Dependent Mechanism.

Gestational diabetes mellitus (GDM) is a disease that changes the function of microvascular of placenta. MicroRNA (miRNA) expression in placenta may contribute to the pathogenesis of GDM. Here, we evaluate the role and function of miR-29b in the development of GDM. This study discovered that miR-29b expression was lower in placentas derived from patients with GDM than that in control placentas. MiR-29b over-expression inhibited cell growth and migration, and miR-29b knockdown promoted cell migration. Then we predicted and confirmed that HIF3A was a direct target of miR-29b with two specific binding sites at the recognition sequences of miR-29b in 3'-UTR of HIF3A mRNA, which was negatively correlated with miR-29b expression level. The up-regulation of HIF3A partially antagonized the inhibitory effect of miR-29b over-expression on cell growth and migration. The enhancement of cell migration induced by miR-29b knockdown was attenuated by down-regulating HIF3A. These results imply that down-regulation of miR-29b may be related with the development of GDM partially via increasing the expression of HIF3A, which may provide a new insight for the mechanism of GDM.

Choline, not folate, can attenuate the teratogenic effects ofdibutyl phthalate (DBP) during early chick embryo development.

Dibutyl phthalate (DBP), a persistent environmental pollutant, can induce neural tube abnormal development in animals. The possible effects of DBP exposure on human neural tube defects (NTDs) remain elusive. In this study, the distribution of DBP in the body fluid of human NTDs was detected by GC-MS. Then, chick embryos were used to investigate the effects of DBP on early embryonic development. Oxidative stress indicators in chick embryos and the body fluid of human NTDs were detected by ELISA. The cell apoptosis and total reactive oxygen species (ROS) level in chick embryos were detected by whole-mount TUNEL and oxidized DCFDA, respectively. The study found that the detection ratio of positive DBP and its metabolites in maternal urine was higher in the NTD population than that in normal controls. 8-hydroxy-2 deoxyguanosine (8-OHDG) and malondialdehyde (MDA) were evidently upregulated and superoxide dismutase (SOD) was observably downregulated in amniotic fluid and urine. Animal experiments indicated that DBP treatment induced developmental toxicity in chick embryos by enhancing the levels of oxidative stress and cell apoptosis. MDA was increased and SOD was decreased in DBP-treated embryos. Interestingly, the supplement of high-dose choline (100 µg/µL), not folic acid, could partially restore the teratogenic effects of DBP. Our data collectively suggest that the incidence of NTDs is closely associated with DBP exposure. This study may provide new insight for NTD prevention.

[Bacterial culture of donor semen: Analysis of results].

OBJECTIVE: To investigate bacterial infection and the distribution of different bacterial species in the donor semen and the influence of different bacterial counts on semen quality. METHODS: Bacterial colonies in the semen samples from 1 126 donors were counted with the Synbiosis Protocol 3 Automatic Colony Counter and the bacterial species with a colony count ≥104 cfu/ml identified with the VITEK2 Compact Automatic Biochemical Analyzer. The Makler Sperm Counting Board was used to examine the semen quality of the semen samples with a colony count = 0 cfu/ml (n = 22, group A), those with a colony count <104 cfu/ml (n = 22, group B) and those with a colony count ≥104 cfu/ml (n = 22, group C). Univariate analysis was employed for comparison of semen quality among different groups. RESULTS: Among the 1 126 donor semen samples cultured, 5 (0.44%) showed mixed bacterial contamination and 993 (88.58%) showed none but with growth of a certain species of bacteria, 2.22% (22/993) with a colony count ≥104 cfu/ml, mainly including Streptococcus bovis, tiny bacilli, Staphylococcus epidermis, and Staphylococcus aureus, among which gram-positive and gram-negative bacteria accounted for 95.45% (21/22) and 4.54% (1/22), respectively. Compared with group A, groups B and C manifested significantly reduced total sperm count (ï¼»567.5 ± 327.6ï¼½ vs ï¼»421.9 ± 155.9ï¼½ and ï¼»389.9 ± 110.6ï¼½ × 106 per ejaculate, P <0.05) and percentage of progressively motile sperm (ï¼»65.0 ± 6.5ï¼½ vs ï¼»61.0 ± 3.5ï¼½ and ï¼»61.6 ± 4.3ï¼½ %, P <0.05). There were no statistically significant differences among the three groups in the semen liquefaction time, semen pH value, total sperm motility or percentage of morphologically normal sperm (P > 0.05). Of the 284 randomly selected semen samples, 34 (11.97%) were found positive for Ureaplasma urealyticum (UU) and no significant difference was observed in the semen quality between the UU-positive and UU-negative samples (P> 0.05). CONCLUSIONS: The bacteria-positive rate is high in the donor semen and the bacterial species are varied, mainly including gram-positive bacteria. Semen quality is reduced with the increased number of bacterial colonies.

Five Novel COL7A1 Gene Mutations in Three Chinese Patients with Recessive Dystrophic Epidermolysis Bullosa.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is an inherited skin disorder with variable severity and heterogeneous genetic involvement. Recessive DEB (RDEB) is a rare heritable blistering skin condition caused by loss-of-function mutations in the COL7A1 gene. AIM: This study aimed to determine the genetic basis of three Chinese RDEB patients from different families and identify correlations between phenotype and genotype. METHODS: All three patients were diagnosed with RDEB based on typical phenotype. Genomic DNA from both the patients and parents was subjected to amplification of all 118 exons and flanking intron boundaries of the COL7A1 gene, followed by Sanger sequencing. RESULTS: Sanger sequencing identified six mutations in the three patients: five novel mutations (c.2321_2322insCTGA in exon 18, c.5924-5927delAACG in exon 72, c.4871delC in exon 53, c.8278G>A in exon 111, and c.1A>G in exon 1) and one recurrent mutation (c.8038G>A in exon 108). The first three novel mutations resulted in a premature termination codon (PTC), the remaining two novel mutations caused a glycine substitution and a loss of the primary ATG start codon, respectively. All patients had a heterozygous PTC mutation combined with either a glycine substitution mutation in the critical triple-helical collagenous domain or a loss of the primary ATG start codon. CONCLUSION: Our findings expand the mutation spectrum of COL7A1 leading to RDEB and confirm that the RDEB phenotype correlates with the compound heterozygous PTC mutation with a missense mutation. This study will aid the molecular diagnosis, genetic counseling and prognosis prediction of RDEB patients.

Involvement of hypoxia-inducible factor-1α in the oxidative stress induced by advanced glycation end products in murine Leydig cells.

Hyperglycemia increases the formation of advanced glycation end products (AGEs), triggers oxidative impairments and influences inducible factor (HIF)-1α protein levels and transactivation function. Compromised HIF-1α in testis leads to male infertility. The aim of the study was to investigate the role of HIF-1α in oxidative stress induced by AGEs in murine Leydig TM3 cells. TM3 cells were treated with 50 µg/ml of AGEs, or HIF-1α siRNA or 500 µM of DMOG (dimethyloxalylglycine) respectively. The cells were also pretreated with HIF-1α siRNA or 500 µM of DMOG and then were treated with 50 µg/ml of AGEs. The formation of reactive oxygen species (ROS) and cell apoptosis was evaluated. The expression of caspase-3, Heme oxygenase (HO)-1, steroidogenic acute regulatory protein (StAR) and cytochrome P450 17α polypeptide 1 (CYP17A1) was examined by Western blotting. AGEs increased ROS production, induced apoptosis and activated HIF-1α and HO-1 in TM3 cells. HIF-1α attenuated the AGE-induced ROS formation and promoted apoptosis via the upregulation of caspase-3. Knockdown of HIF-1α inhibited the expression of CYP17A1 and StAR, and enhanced the inhibition of StAR and CYP17A1 by AGEs. These findings indicate that attenuated HIF-1α exacerbates the oxidative stress injury by AGEs in murine Leydig cells, and contributes to diabetic male infertility.

Effects of atrazine on the oxidative damage of kidney in Wister rats.

The environmental persistence and bioaccumulation of herbicide atrazine may pose a significant threat to human health. In this experiment, 4 weeks old female Wister rats were treated by 0, 5, 25 and 125 mg/kg atrazine respectively for 28 days, and the oxidative stress responses as well as the activations of Nrf2 signaling pathway in kidney tissues induced by atrazine were observed. The results showed that after be treated by atrazine, the Blood urea nitrogen (BUN) and creatinine (CREA) levels in serum were increased, the contents of nitric oxide (NO) and malondialdehyde (MDA) in the kidney tissue homogenates were increased, the over-expressed Nrf2 transferred into the nuclei and played an antioxidant role by up-regulated the expression of II phase detoxifying enzymes such as heme oxygenase-1 (HO1) and NAD(P)H quinone oxidoreductase (NQO1) and the expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).

The dynamics of polycomb group proteins in early embryonic nervous system in mouse and human.

Polycomb group (PcG) proteins are transcription regulatory proteins that control the expression of a variety of genes and the antero-posterior neural patterning from early embryogenesis. Although expression of PcG genes in the nervous system has been noticed, but the expression pattern of PcG proteins in early embryonic nervous system is still unclear. In this study, we analyzed the expression pattern of PRC1 complex members (BMI-1 and RING1B) and PRC2 complex members (EED, SUZ12 and EZH2) in early embryonic nervous system in mouse and human by Western blot and Immunohistochemistry. The results of Western blot showed that EED protein was significantly up-regulated with the increase of the day of pregnancy during the early embryogenesis in mouse. BMI-1 protein level was significantly increased from the day 10 of pregnancy, when compared with the day 9 of pregnancy. But the SUZ12, EZH2 and RING1B protein level did not change significantly. From the results of Immunohistochemistry, we found that the four PcG proteins were all expressed in the fetal brain and fetal spinal cord in mouse. In human, the expression of EED, SUZ12, and EZH2 was not significantly different in cerebral cortex and sacral spinal cord, but BMI-1 and RING1B expression was enhanced with the development of embryos in early pregnancy. Collectively, our findings showed that PRC1 and PRC2 were spatiotemporally expressed in brain and spinal cord of early embryos.

Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos.

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10⁻³ M MBP impaired developmental competency, whereas exposure to 10⁻4 M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10⁻³ M MBP. In addition, 10⁻³ M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10⁻5, 10⁻4 and 10⁻³ M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

A network of miRNAs expressed in the ovary are regulated by FSH.

The process of folliculogenesis requires a tightly regulated series of gene expression that are a pre-requisite to the development of ovarian follicle. Among these genes, follicle-stimulating hormone (FSH) is notable for its dual role in development of follicles as well as proliferation and differentiation of granulosa cells. The post-transcriptional expression of these genes is under the control of microRNAs (miRNAs), a class of small, endogenous RNAs that negatively impact gene expression. This study was carried out to determine the role of several miRNAs including mir-143, let-7a, mir-125b, let-7b, let-7c, mir-21 in follicular development in the mouse. The expression of these RNAs was very low in primordial follicles but these became readily detectable in the granulosa cells of primary, secondary and antral follicles. We show that this expression of some miRNAs (mir-143, let-7a, mir-15b) is under negative control of FSH. Together, these findings suggest that FSH regulates folliculogenesis by a novel pathway of miRNAs.

Teratogenic effects of sodium thiosulfate on developing zebrafish embryos.

Sulfuric derivatives are potentially hazardous to human health, especially during embryogenesis. Zebrafish were used to study the toxic effect of sodium thiosulfate (STS) (1~1 x 10(-6) mol/L) on embryo development with real-time in vivo imaging. Motor neuron differentiation and proliferation were analyzed by detecting the dynamics of acetylated tubulin (alpha-tubulin) and of proliferating cell nuclear antigen (PCNA). The expression pattern of brain- and muscle-specific microRNAs was detected by whole-mount in situ hybridization. The development of embryos exposed to 0.1~1 mol/L STS was severely retarded and was accompanied by malformation of multiple organs; embryos exposed to 10 micromol/L~10 mmol/L STS had circulatory, nervous and maxillofacial malformations. Embryos were more sensitive to STS at 48 hours post fertilization (hpf) compared with 24 and 96 hpf. STS can destroy the normal development of motor neurons and can affect cell proliferation. We also found differential expression of miR-124a and miR-133a in STS-treated embryos. STS interferes with the normal cytoskeleton structure, inhibits cell proliferation and leads to nervous, cardiac and maxillofacial malformations. MiR-124a and miR-133a were involved in STS malformation induction.

Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos.

Arsenic usually accumulates in soil, water and airborne particles, from which it is taken up by various organisms. Exposure to arsenic through food and drinking water is a major public health problem affecting some countries. At present there are limited laboratory data on the effects of arsenic exposure on early embryonic development and the mechanisms behind its toxicity. In this study, we used zebrafish as a model system to investigate the effects of arsenite on early development. Zebrafish embryos were exposed to a range of sodium arsenite concentrations (0-10.0mM) between 4 and 120h post-fertilization (hpf). Survival and early development of the embryos were not obviously influenced by arsenite concentrations below 0.5mM. However, embryos exposed to higher concentrations (0.5-10.0mM) displayed reduced survival and abnormal development including delayed hatching, retarded growth and changed morphology. Alterations in neural development included weak tactile responses to light (2.0-5.0mM, 30hpf), malformation of the spinal cord and disordered motor axon projections (2.0mM, 48hpf). Abnormal cardiac function was observed as bradycardia (0.5-2.0mM, 60hpf) and altered ventricular shape (2.0mM, 48hpf). Furthermore, altered cell proliferation (2.0mM, 24hpf) and apoptosis status (2.0mM, 24 and 48hpf), as well as abnormal genomic DNA methylation patterning (2.0mM, 24 and 48hpf) were detected in the arsenite-treated embryos. All of these indicate a possible relationship between arsenic exposure and developmental failure in early embryogenesis. Our studies suggest that the negative effects of arsenic on vertebrate embryogenesis are substantial.

[A novel PAX6 mutation (c.1286delC) in the patients with hereditary congenital aniridia.].

To study the molecular genetic mechanism of hereditary congenital aniridia, the entire coding exons (exon 4-13) of PAX6 gene and the flanking exon-intron junctions were amplified through PCR from the genomic DNA of all the two patients in a Chinese family with aniridia. PCR products were purified from agarose gel and sequenced. In both patients, a novel deletion mutation (c. 1286delC) in exon 11 was identified. Compared with the normal product of PAX6 gene, this mutation caused frame shifting, and generated a novel 55 amino acid peptide from codon 309. This deletion also resulted in a premature termination codon (PTC) and preterminated peptide synthesis. Meanwhile, this mutation was absent in all the unaffected family members and 50 normal control individuals through PCR-RFLP.

Nordihydroguaiaretic acid restores expression of silenced E-cadherin gene in human breast cancer cell lines and xenografts.

In our study we use nordihydroguaiaretic acid (NDGA), the naturally occurring lignan, to investigate whether it plays a role in the prevention and treatment of cancer by epigenetic modifications. The growth inhibitory effect of NDGA on human breast cancer cell lines was determined using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). It substantially inhibited the growth of human breast cancer cell lines SKBR3 and MDA-MB-435 with an estimated IC50 of 31.09+/-1.6 and 38.8+/-2.1 micromol/l respectively, after 4 days incubation with different NDGA concentrations. The in-vivo anticancer activity of NDGA was evaluated by calculating the tumor growth inhibition value. NDGA substantially inhibited the growth of human breast carcinoma cells in both animal and cell-based models. We also found that a single treatment with NDGA reactivates methylation-silenced E-cadherin gene in vitro and in vivo, suggesting an intriguing concept that lignans may act as natural effective epigenetic modifiers in the prevention and treatment of cancer.

Reactivation of methylation-silenced tumor suppressor gene p16INK4a by nordihydroguaiaretic acid and its implication in G1 cell cycle arrest.

Phytoestrogens, including the two major groups isoflavones and lignans, are chemicals with weak estrogenic activity which occur naturally in many foods and herbs. Recently, several intriguing studies reported that some isoflavones can affect DNA methylation status. However, little is known about the effect of plant lignans on epigenetic modification. Using cultured T47D and RKO human cancer cells as a model, we studied the modulating effects of nordihydroguaiaretic acid (NDGA), a member of the lignan family, on the methylation status of the gene promoter region. Our results indicated that NDGA reverses p16INK4a CpG island hypermethylation, and restores its transcription and expression in both cell lines. Cytometric analysis showed that NDGA significantly affects cell cycle progression by arresting cells at the G1 phase. Consistent with the reacquisition of p16INK4a expression, we also found that NDGA induces cellular senescence in cancer cells. This is the first study demonstrating that a member of the lignan family can induce demethylation in human cancer cell lines, suggesting a novel epigenetic mechanism in the prevention or treatment of cancer.

Retinoic acid downregulates microRNAs to induce abnormal development of spinal cord in spina bifida rat model.

OBJECTS: MicroRNAs have been found in the developing central nervous system, but little is known about their functions in development, especially in the abnormal development of spinal cord in spina bifida. To this end, we have studied the mechanism of microRNAs involved in the morphogenesis of the spinal cord in all-trans-retinoic acid (RA)-treated spina bifida rat fetus. MATERIALS AND METHODS: Timed-pregnant rats were gavage-fed RA, and embryos were obtained on 13.5, 15.5, 17.5, and 19.5 days. MicroRNAs' expression profile was analyzed by Northern blot. In situ apoptosis detection and microRNA in situ hybridization methods on sections of paraffin-embedded tissues were employed to explore the mechanism. CONCLUSION: Administration of RA reduced the size of the spinal cord, probably as a consequence of increased cell death. There is a dramatic decrease in the expression of miR-9/9*, miR-124a and miR-125b, and Bcl2 and P53 as well in the sacral cord from E13.5 to E19.5 days post coitum. Our data showed that expression of these microRNAs was dysregulated in RA-treated spinal cord during embryonic development, suggesting that they may be involved in the development of the spinal cord.

Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray.

OBJECTS: Our objective was to develop an oligonucleotide DNA microarray (OMA) for genome-wide microRNA profiling and use this method to find miRNAs, which control organic development especially for nervous system. MATERIALS AND METHODS: Eighteen organic samples included cerebrum and spinal cord samples from two aborted human fetuses. One was 12 gestational weeks old (G12w) and the other was 24 gestational weeks old (G24w). Global miRNA expression patterns of different organs were investigated using OMA and Northern blot. CONCLUSION: The OMA revealed that 72-83% of miRNAs were expressed in human fetal organs. A series of microRNAs were found specifically and higher-expressed in the human fetal nervous system and confirmed consistently by Northern blot, which may play a critical role in nervous system development.