miR-20a-5p promotes pulmonary artery smooth muscle cell proliferation and migration by targeting ABCA1.

BACKGROUND: The function of miR-20a-5p in pulmonary artery smooth muscle cells (PASMCs) and the underlying mechanism remains largely unknown. METHODS: C57BL/6J mice and PASMCs were used for constructing pulmonary artery hypertension (PAH) animal and cell models, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect miR-20a-5p and ATP-binding cassette subfamily A member 1 (ABCA1) messenger RNA expression. CCK-8, Transwell, and TUNEL experiments were used to determine PASMCs proliferation, migration, and apoptosis. The relationship between miR-20a-5p and ABCA1 was detected by luciferase reporter experiment, Western blot analysis, and qRT-PCR. RESULTS: miR-20a-5p was remarkably elevated in PASMCs of PAH mice and human PASMCs treated by hypoxia, while ABCA1 was remarkably decreased. After transfection of miR-20a-5p mimics, PASMCs proliferation and migration were promoted and PASMCs apoptosis was suppressed. ABCA1 was confirmed to be a target of miR-20a-5p and restoration of ABCA1 reversed the function of miR-20a-5p. CONCLUSION: miR-20a-5p enhances the proliferation and migration of PASMCs to promote the development of PAH via targeting ABCA1.

The epigenetic modifications and the anterior to posterior characterization of meiotic entry during mouse oogenesis.

The meiotic initiation of mammalian oogonia is a critical step during the development of primordial germ cells (PGCs) to mature oocytes. In this study, a systematic investigation of epigenetic modifications and DAZL gene expression during oogonia meiotic entry were performed. We found that the expression of DAZL was epigenetically regulated by DNA methylation of CpG islands within its promoter region. During meiotic entry, a continuously increasing level of 5hmC, a stable epigenetic marker usually associated with the activation of gene expression, was observed from 11.5 to 16.5 dpc (days post coitum). Meanwhile trimethylation of lysine 27 on histone3 (H3K27me3), usually associated with repression of gene expression, had a sustainable increase from 12.5 to 16.5 dpc. Finally, by equally dividing the ovaries into three regions representing the anterior, the middle, and the posterior of the ovary and performing immunofluorescence and qRT-PCR on the individual regions, we provided further evidences that the meiotic entry and progression of female germ cells is in an anterior to posterior pattern.

Interaction of the transforming growth factor-ß and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-ß signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-ß signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-ß signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-ß signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-ß and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

Insulin regulates primordial-follicle assembly in vitro by affecting germ-cell apoptosis and elevating oestrogen.

Insulin is a protein secreted by pancreatic ß-cells, which plays an important role in the regulation of ovarian function. However, the specific molecular mechanism of its function remains largely unknown. This study aimed to assess the effect of insulin on mouse folliculogenesis using an in vitro ovary-culture model. The results demonstrated that insulin promoted the proliferation of ovarian granulosa cells in vitro, and thereby accelerated the progress of folliculogenesis (the percentage of oocytes in cysts declined from 42.6% to 29.3%); however, the percentage of apoptotic oocytes increased after insulin treatment. Further investigation indicated that apoptosis occurred mainly in germ-cell cysts. After 3 days of insulin treatment, oestrogen in the culture medium of mouse ovaries significantly increased (P<0.01), while the lower dose of oestrogen promoted primordial-follicle assembly in vitro. In conclusion, insulin promoted folliculogenesis by facilitating germ-cell apoptosis within the cysts and upregulating oestrogen levels.

Trehalose 6-phosphate phosphatase is required for development, virulence and mycotoxin biosynthesis apart from trehalose biosynthesis in Fusarium graminearum.

Trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) are required for trehalose biosynthesis in yeast and filamentous fungi, including Fusarium graminearum. Three null mutants Δtps1, Δtps2 and Δtps1-Δtps2, each carrying either a single deletion of TPS1 or TPS2 or a double deletion of TPS1-TPS2, were generated from a toxigenic F. graminearum strain and were not able to synthesize trehalose. In contrast to its reported function in yeasts and filamentous fungi, TPS1 appeared dispensable for development and virulence. However, deletion of TPS2 abolished sporulation and sexual reproduction; it also altered cell polarity and ultrastructure of the cell wall in association with reduced chitin biosynthesis. The cell polarity alteration was exhibited as reduced apical growth and increased lateral growth and branching with increased hyphal and cell wall widths. Moreover, the TPS2-deficient strain displayed abnormal septum development and nucleus distribution in its conidia and vegetative hyphae. The Δtps2 mutant also had 62% lower mycelial growth on potato dextrose agar and 99% lower virulence on wheat compared with the wild-type. The Δtps1, Δtps2 and Δtps1-Δtps2 mutants synthesized over 3.08-, 7.09- and 2.47-fold less mycotoxins, respectively, on rice culture compared with the wild-type. Comparative transcriptome analysis revealed that the Δtps1, Δtps2 and Δtps1-Δtps2 mutants had 486, 1885 and 146 genotype-specific genes, respectively, with significantly changed expression profiles compared with the wild-type. Further dissection of this pathway will provide new insights into regulation of fungal development, virulence and trichothecene biosynthesis.

Effects of diethylhexyl phthalate (DEHP) given neonatally on spermatogenesis of mice.

We previously demonstrated that the effects of diethylhexyl phthalate (DEHP) alter reproduction function on male mice. Immature male mice were treated daily with DEHP from postnatal day 7-21, 7-35, 7-49, in a dose-dependent manner. As results, both the quality and quantity of spermatozoa were decreased in 60-day-old mice. The results by RT-PCR analysis indicated that DDx3Y, Usp9Y, RBM, E1F1AY, EGF, FSHR and EGFR genes were down-regulated, and LHR, Cyp17a1 and Cyp19a1 were down-regulated in response to DEHP. These genes were selected based on their markedly increased or decreased expression levels. However, DEHP had no effect on the meiotic process and recombination levels in male mouse germ cells. Treatment with DEHP induced histopathological changes in the testes. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of DEHP in humans and wildlife.

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells.

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 µg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.

Polysaccharide extracted from Huperzia serrata using response surface methodology and its biological activity.

In this study, Huperzia serrata polysaccharide (HSP) fraction was isolated using response surface methodology (RSM) and Box-Behnken design (BBD). The extraction time, temperature and ratio of water to raw material were employed effects. And properties of four polysaccharide (60%-HSP, 70%-HSP, 80%-HSP and 90%-HSP) were evaluated. The results indicated that the optimal extraction conditions were the following: 3.07 h, 49.46 °C and a liquid material ratio of 20.73:1. The four HSP presented irregular aggregation of shape. And all HSP exhibited antioxidant and anticancer activities.

Extraction of polysaccharides from Codonopsis pilosula by fermentation with response surface methodology.

Codonopsis pilosula is a kind of traditional Chinese medicine used to treat weak spleens, stomach problems, anemia, and fatigue. Polysaccharide is one of main components of Codonopsis pilosula. In this study, response surface methodology (RSM) was used to optimize the extraction parameters of Codonopsis pilosula polysaccharides (CPP) by fermentation. The exaction temperature (°C), yeast liquid volume (2 mg/ml, ml), and time (h) were employed effects. Results indicated that the best extraction conditions were the following: extraction temperature 24.75°C, yeast liquid volume 2.96 ml (5.92 mg), and a fermentation time of 21.03 hr. After purification with DE52 and Sephadex G-100, the molecular structure was determined by ultraviolet-visible (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) (1H and 13C). The monosaccharide composition of CPP1 was determined to be mannose (1.76%), glucose (97.38%), and arabinose (0.76%). CPP1 exhibited high antioxidant activities in scavenging ABTS radicals, ferreous ions, and superoxide ion radicals. Thus, CPP1 could be used as an antioxidant or functional food.

Nicotine exposure impairs germ cell development in human fetal ovaries cultured in vitro.

In the present paper, we found that human fetal ovaries (at ~16 weeks) express the transcripts for several subunits of the nicotinic acetylcholine receptor (nAChR). Exposure to the drug in vitro resulted in the marked increase of apoptosis in the ovaries in a time and dose-dependent manner. Evidence that adverse nicotine effects are potentially due to an increased level of reactive oxygen species (ROS) and consequent DNA damage, both in the ovarian somatic cells and germ cells, are reported. After 4 days of culture, exposure to 1 mM and 10 mM nicotine caused a 50% and 75% decrease, respectively, in the number of oogonia/oocytes present in the fetal ovaries. These results represent the first indication that nicotine may directly cause apoptosis in cells of the fetal human ovary and may lead to a reduction of the ovarian reserve oocytes and consequent precocious menopause in mothers smoking during pregnancy.

Tissue-Specific Regulation of the Contents and Correlations of Mineral Elements in Hens by Zinc Oxide Nanoparticles.

Due to their small size, zinc oxide (ZnO) nanoparticles (NPs) are readily absorbed and easily cross biological barriers, which make them promising candidates as diet additives. However, some studies have reported that ZnO NPs cause toxicity; therefore, their safety and potency as diet additives for farm animals should be established. This study was the first to fully evaluate the effects of ZnO NPs on the homeostasis of eight elements in seven organs/tissues. The regulation of element homeostasis was found to be organ specific with no influence on oxidation status, anti-oxidation capability, or organ damage. ZnO NPs may specifically regulate the homeostasis of mineral elements and affect the following correlations: (1) between the element content in each organ and the concentration of Zn used in ZnSO4 or ZnO NP treatments; (2) between ZnO NP and ZnSO4 treatments for the same element in each organ; and (3) between elements (in each organ in ZnSO4 or ZnO NP treatments) in layers' organs/tissues. The use of ZnO NPs as diet additives for animals should be implemented cautiously because, among other uncertainties, they may affect mineral element content.

Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty.

Diethylhexyl phthalate exposure impairs follicular development and affects oocyte maturation in the mouse.

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a commercial plasticizer and present in medical devices, tubing, food containers and packaging. It is considered an endocrine disruptor and studies on experimental animals showed that exposure to DEHP can alter the function of several organs including liver, kidneys, lungs and reproductive system, particularly the developing testes of prenatal and neonatal males. Exposure to DEHP has been proposed as a potential human health hazard. This study assessed the effects of DEHP on folliculogenesis and oocyte maturation using the mouse as the experimental model. Newborn female mice were hypodermically injected with DEHP at doses of 20 and 40 µg/kg per body weight following different exposure regimens during the weaning period. We found that DEHP altered both folliculogenesis and oocyte development. In particular, DEHP exposure significantly decreased the number of the primordial follicles at pubertal and adult age by possibly accelerating the rate of follicle recruitment dynamics, reduced and/or delayed the level of imprinted gene methylation in the oocytes and increased metaphase II spindle abnormalities in oocytes matured in vitro. Furthermore, the weight of pups and litter size of mothers exposed to DEHP were significantly lower than controls. Finally, the number of primordial follicles appeared significantly reduced also in the F1 offspring at the adult age. These results show that DEHP may have a number of adverse effects on oogenesis, especially when exposure occurs during early postnatal age, arising concerns about the exposure of human female infants and children to this compound.

Expression and epigenetic dynamics of transcription regulator Lhx8 during mouse oogenesis.

The spatial and temporal specific activation and inhibition of numerous genes are required for successful oogenesis which is precisely regulated by germ cell-related transcription factors, and appropriate epigenetic modifications, including DNA methylation, histone modification and other mechanisms that closely regulate the functional exertion of these transcription factors. In this study, we characterized the correlation between the expression and epigenetic dynamics of Lhx8, a germ cell specific transcription factor during mouse oogenesis. Immunohistochemistry, quantitative PCR and western blots were performed to localize and quantify the expressional characteristics of Lhx8 in oocytes of 13.5 dpc (day post coitum), 17.5 dpc, 0 dpp (day post partum), 3 dpp, 7 dpp and 14 dpp. The results showed that LHX8 protein was located in the nucleus of oocytes, and increasingly expressed during primordial follicle activation. Sequencing of bisulfite-converted genomic DNAs revealed that the methylation dynamics of Lhx8-3' was highly changeable but almost no change occurred in Lhx8-5'. ChIP-QPCR analysis showed that histone H3 acetylation of Lhx8 was also increased during primordial follicle assembly and activation. In conclusion, Lhx8 expression is related with the activation of primordial follicles, which is highly correlated with the demethylation of Lhx8-3' untranslated region and the high acetylation of histone H3.