•TGP profiles were established by HPSEC.•Vegetable oils and waste cooking oils were differentiated by the difference on TGP contents.•TGP showed an application potential on the identification of cold pressed or refined oils.•The determination of TGP content could be an effective way for quality evaluation of vegetable oils.The stability and low volatility of triacylglycerol polymer (TGP) makes it a reliable index for evaluating the oxidative levels of vegetable oils. The present work established the TGP profiles for 12 different types of vegetable oils commonly observed in the market and 44 waste cooking oil samples by high-performance size-exclusion chromatography (HPSEC) analysis. The TGP contents of vegetable oils ranged from 0.10% to 1.67%, while those of waste cooking oils were between 3.18% and 18.75%. Moreover, the TGP contents were applied for differentiating vegetable and waste cooking oils, and vegetable oils were processed by cold pressed and refined technology. The results from this study demonstrated that TGP quantification had a promising prospect for the quality evaluation of vegetable oils.

•MC-LR could disrupt cell proliferative and increase cell apoptosis in the testis of the male offspring.•The exposure to MC-LR resulted in an altered piRNA expression profile.•This study found an important link between piRNA and MC-LR-induced reproductive system toxicity.In the present study, we evaluated the toxic effects on the testis of the male offspring of MC-LR exposure during fetal and lactational periods. Pregnant females were distributed into two experimental groups: control group and MC-LR group which were exposed to 0 and 10 μg/L of MC-LR, respectively, through drinking water separately during fetal and lactational periods. At the age of 30 days after birth, the male offspring were euthanized. The body weight, testis index, and histomorphology change were observed and the global changes of piwi-interacting RNA (piRNA) expression were evaluated. The results revealed that MC-LR was found in the testis of male offspring, body weight and testis index decreased significantly, and testicular tissue structure was damaged in the MC-LR group. In addition, the exposure to MC-LR resulted in an altered piRNA expression profile and an increase of the cell apoptosis and a decrease of the cell proliferation in the testis of the male offspring. It was reasonable to speculate that the toxic effects on reproductive system of the male offspring in MC-LR group might be mediated by piRNAs through the regulation of the target genes. As far as we are aware, this is the first report showing that MC-LR could play a role in disorder of proliferative and cell apoptosis in the testis of the male offspring by the maternal transmission effect of toxicity.Download high-res image (312KB)Download full-size image

•We firstly report MC-LR could impact the expression of miRNAs and mRNAs in mGCs.•We firstly analyzed aberrant expression of miRNAs and mRNAs by bioinformatics in mGCs after MC-LR exposure.•Disorder of miRNAs and mRNAs may affect the hormone production and growth of mGCs.Microcystin is a cyclic heptapeptide compounds which could cause female mammals' reproductive toxicity. Ovarian granulosa cells (GCs) are essential for the growth and development of follicles. In this study, after mouse granulosa cells (mGCs) treated with microcystin-LR (MC-LR) for 48 h, microRNAs (miRNAs) and mRNAs microarray technology were adopted to detect the expression of miRNAs and mRNAs. The results showed that 125 miRNAs and 283 mRNAs changed significantly, including 50 miRNAs down-regulated (fold change < −1.2), 75 miRNAs up-regulated (fold change > 1.2), 162 mRNAs down-regulated (fold change < −1.15) and 121 mRNAs up-regulated (fold change > 1.15) in treated group compared with the control group. Functional analysis showed that significant changed miRNAs and mRNAs are mainly involved in proliferation, apoptosis, immunity, metabolism and other biological processes of mGCs. By KEGG pathways analysis, we found that differentially expressed miRNAs and mRNAs mainly participated in apoptosis, formation of cancer, proliferation, production of hormones and other related signal pathways. miRNA-gene network analysis indicated that miR-29b-3p, miR-29a-3p, miR-29c-3p, miR-1906, miR-182-5p, growth factor receptor bound protein 2-associated protein 2 (Gab2), FBJ osteosarcoma oncogene (Fos), insulin-like growth factor 1 (Igf1), mannosidase 1, alpha (Man1a) are key miRNAs and genes. The microarray results were validated by real-time fluorescent quantitative PCR (qRT-PCR).

Familial aggregation of cancer may reflect an overall contribution of inherited genes or a shared mechanism for the manipulation of gene function. DNA methylation in the promoter regions is considered to be a mechanism through which tumor suppressor genes are inhibited, which will lead to tumorigenesis and tumor progression. To evaluate the association between the methylation status in the promoter of estrogen receptor (ER) β,possibly a tumor suppressor gene specific for prostate cancer, and the risk in prostate cancer in a Chinese population, a case–control study that included 56 sporadic prostate cancer cases and 60 healthy controls was conducted. Genomic DNA was extracted from peripheral blood of all the subjects for analyzing the methylation status of the ERβ promoter by methylation-specific PCR, which was verified by bisulfite genomic sequencing PCR. A significant difference was observed in the methylation frequencies of the ERβ promoter between cancer patients (12/56, 21.4 %) and healthy controls (5/60, 8.3 %). Prostate cancer (PC-3 and DU-145) and prostatic epithelial (RWPE-1) cell lines were treated with various concentrations of the methyltransferase inhibitor 5-Aza-2′-dC. Expression of ERβ was detected at both transcriptional and translational levels. As a result, both mRNA and protein of ERβ were elevated following treatment with increasing concentrations of the demethylating agent. Taken together, our results support the conclusion that abnormal methylation of the ERβ promoter may increase genetic susceptibility to prostate cancer.

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin, especially by decreasing sperm quality through affecting spermatogonia. However, the molecular mechanisms of MC-induced spermatogonial cytotoxicity still remain unclear. The present study was designed to investigate changes in microRNA (miRNA) profiles and their potential functions in spermatogonia (GC-1 cell line) following treatment with MC-LR. With microarray analysis, 101 miRNAs were identified to be significantly altered in GC-1 cells treated with MC-LR. Among the 25 miRNAs associated with spermatogenesis, miR-96 was down-regulated most dramatically and thus selected for further functional analysis. Deleted-in azoospermia-associated protein 2 (DAZAP2) was predicted to have a binding sequence for miR-96 within its 3′-untranslated region. Fluorescent reporter assay confirmed that DAZAP2 was the target gene of miR-96. The expression of DAZAP2 decreased significantly when miR-96 was up-regulated. Consistently, down-regulation of miR-96 significantly increased the level of DAZAP2. Up-regulation of miR-96 promoted cell viability in GC-1 cells as a result of exposure to MC-LR. Our study suggested a crucial role for miR-96 in the regulation of cytotoxic effects of MC-LR in spermatogonia, which provides new perspectives in the diagnosis and treatment strategies for MC-induced male infertility.

Microcystins (MCs) are a group of cyclic heptapeptide toxins produced by naturally freshwater cyanobacteria. Among more than 90 identified analogues of microcystins, microcystin-LR (MC-LR) is the most abundant and toxic. Our previous investigations indicated that MC-LR displays male reproductive toxicity, but the target of MC-LR in testes remains unclear. To this end, the present study is designed to elucidate whether microcystin-LR could be distributed to testes and explore the target cells in testes. In the in vivo study, male Sprague–Dawley rats were injected intraperitoneally with MC-LR at a dose of 300 μg/kg per day for 6 days. MC-LR was detected in testes, mainly within seminiferous tubules, which was further validated by Western blot. The concentrations of MC-LR were determined by LC–MS analysis, with a result of 0.0252 ± 0.0037 and 0.0056 ± 0.0012 μg/g dry weight in liver and testis respectively. In the in vitro study, Primary cultured spermatogonia, Sertoli cells and Leydig cells were exposed to MC-LR respectively, and MC-LR was observed to enter spermatogonia and Sertoli cells, but not Leydig cells. These results suggested that the reproductive toxicity of MC-LR were induced by its distribution in testis. Spermatogonia and Sertoli cells are important target cells.

Nonylphenol (NP) is a common environmental contaminant that is known to disrupt the reproductive system. The testicular Sertoli cells play a pivotal role in the regulation of spermatogenesis and are susceptible to NP-induced reproductive lesions. Our goal was to ascertain whether NP could induce apoptosis in Sertoli cells and to explore the preapoptotic changes in Sertoli cells at low NP concentrations, similar to environmental conditions. In order to survey events that occur at the protein level in Sertoli cells after exposure to NP, we used a proteomic approach with two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify proteins with altered expression in rat Sertoli cells treated with 0.01 and 0.1 μM NP for 24 h. We separated 63 protein spots and identified 41 that were differently expressed in the NP-treated groups and the control. Of these 41 spots, we focused on Raf kinase inhibitor protein (RKIP), Annexin A7 (ANXA7), ERp57, and Peroxiredoxin 6 (PRDX6) for further analysis by Western blot. These proteins are involved in the response of Sertoli cells to programmed cell death. These data help to outline mechanisms by which NP might induce apoptotic tendencies in Sertoli cells.

The herbal immunoregulation mixture (HIRM) were extracts of several traditional Chinese medicines (TCMs): Astragalus membranaceus (from the root and stem), Polygonum multiflorum Thunb. (from the root), Isatis tinctoria L. (from the root), Glycyrrhiza glabra (from the stem). Immune parameters, which included macrophage phagocytic activity, macrophage reactive oxygen species (ROS), activity of serum lysozyme, nitric oxide synthetase (NOS) and superoxide dismutase (SOD), levels of total serum protein, globulin, albumin, triglyceride and cholesterol, were determined in carp that had been fed diets containing HIRM at 0.5% or 1% for 30 days. The results showed that, compared with those in the control group, the diets with 0.5% and 1% HIRM resulted in significant increase in macrophage phagocytic activity, macrophage ROS and the levels of total protein, globulin, albumin and NOS activity in serum (P < 0.05), and no significant difference was found in SOD, lysozyme activities and triglyceride level (P > 0.05). On the other hand, 0.5% HIRM led to evident enhancement of NOS activity and cholesterol level compared to 1% HIRM. These results indicated that HIRM might elevate the function of immunity in carp (Cyprinus carpio).

The effects of chronic low-dose exposure to microcystins were preliminarily studied on sperm quality and testicular function in male mice. Microcystin-LR (MC-LR) was orally administered to male mice at 0, 1, 3.2, and 10 μg/L for 3 and 6 months. Our preliminary study found in three-month group, sperm quality declined at 3.2 and 10 μg/L doses, testosterone dropped at 10 μg/L, levels of LH and FSH increased, and Leydig cells exhibited apoptosis. Similar, but more pronounced, effects were observed in groups treated with MC-LR for 6 months. Compared to control (0 μg/L), the rate of sperm abnormality was higher and testosterone levels were lower following administration of 3.2 and 10 μg/L MC-LR and structural damage to the testis was observed with 10 μg/L dose. Thus, chronic low-dose treatment with MC-LR results in substantial toxicity to male reproduction, causing declines in sperm quality, decreased levels of serum testosterone, and injury to the testis.

The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EEDs) which at high doses in some species of laboratory animals have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EEDs in the environment, their combined effects warrant investigation. In this study, we attempted to clarify the interactions of NP and DBP on tight junctions (TJs) between rat Sertoli cells. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from Sprague-Dawley rats, and treated with NP and MBP, singly or combined. The morphology of Sertoli cells, and structure and functionality of TJs were measured. In the in vivo experiment, rats were gavaged on postnatal day 23–35 with a single or combined NP and DBP treatment. Testicular weight and morphology of TJs were recorded. These data indicated that NP and DBP/MBP, either in single or in combination, induced the structural and function changes of Sertoli cell tight junctions, both in vivo and in vitro. The combined effect on the regulation of TJ proteins at both the protein and gene levels was correlated to the effect exerted by NP, suggesting that the structure and function of Sertoli cells were more sensitive to exposure to NP than MBP.

The estrogenic chemical nonylphenol (NP) and the antiandrogenic agent di-n-butyl phthalate (DBP) are regarded as widespread environmental endocrine disruptors (EEDs) which at high doses in some species of laboratory animals have adverse effects on male reproduction and development. Given the ubiquitous coexistence of various classes of EEDs in the environment, their combined effects warrant investigation. In this study, we attempted to clarify the interactions of NP and DBP on tight junctions (TJs) between rat Sertoli cells. In the in vitro experiment, monobutyl phthalate (MBP), the active metabolite of DBP, was used instead of DBP. Sertoli cells were isolated from Sprague-Dawley rats, and treated with NP and MBP, singly or combined. The morphology of Sertoli cells, and structure and functionality of TJs were measured. In the in vivo experiment, rats were gavaged on postnatal day 23–35 with a single or combined NP and DBP treatment. Testicular weight and morphology of TJs were recorded. These data indicated that NP and DBP/MBP, either in single or in combination, induced the structural and function changes of Sertoli cell tight junctions, both in vivo and in vitro. The combined effect on the regulation of TJ proteins at both the protein and gene levels was correlated to the effect exerted by NP, suggesting that the structure and function of Sertoli cells were more sensitive to exposure to NP than MBP.

Pulmonary fibrosis is a progressive lung disorder of unknown etiology, which is characterized by alterations in alveolar epithelium function, fibroblast activation, and increased extracellular matrix deposition. Recent studies have demonstrated that PF is associated with uncontrolled production of cytokines after lung injury. In the present study, we found that transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2 (FGF-2) were both upregulated in bleomycin-induced fibrotic lung tissue and primary murine alveolar epithelial Type II (ATII) cells treated with bleomycin. Furthermore, we discovered that TGF-β1 could induce the differentiation of lung resident mesenchymal stem cells (LR-MSCs) into fibroblasts, which may play an essential role in PF. LR-MSCs incubated with FGF-2 showed modest alterations in the expression of α-SMA and Vimentin. Moreover, in our study, we found that Wnt/β-catenin signaling was activated both in vitro and in vivo as a result of bleomycin treatment. Interestingly, we also found that suppression of the Wnt/β-catenin signaling could significantly attenuate bleomycin-induced PF accompanied with decreased expression of TGF-β1 and FGF-2 in vitro and in vivo. These results support that controlling the aberrant expression of TGF-β1 and FGF-2 via inhibition of Wnt/β-catenin signaling could serve as a potential therapeutic strategy for PF.

Nonylphenol (NP) is a widely distributed environment contaminant and has been documented to disrupt testicular development and decrease male fertility. Amongst possible targets of this compound are testicular Sertoli cells, which play a crucial role in supporting and nourishing sperm cells. In the present study, we found that NP treatment could cause dramatic morphological changes as well as decreased cell viability of Sertoli cells, while the following Annexin V–PI staining demonstrated that NP treatment led to increased proportion of cell apoptosis, which was evidenced again by the detection of condensation and marginal changes of chromatins using Hoechst staining and transmission microscopy observation. In addition, increased intracellular Ca2+ levels and changes of endoplasmic reticulum (ER) ultrastructure were also observed in NP-treated groups, indicating the action of NP on ER. The subsequent data showed that the expressions of ER-stress signaling targeted genes GRP78 and gadd153 were elevated, suggesting the activation of ER-stress signal pathway. Furthermore, the detection of ER-stress related proteins by western blotting revealed that the expression of gadd153 was upregulated by NP, whereas the expressions of GRP78 and ERp57 were both first upregulated and then inhibited. Taken together, it is suggested that NP can induce ER stress in Sertoli cells, which may plays an important role in the induction of apoptosis.

•MC-LR could enter Sertoli cells and exhibits cytotoxicity.•Autophagy and apoptosis were both induced with MC-LR treatment in Sertoli cells.•The accumulated autophagosomes will promote the apoptotic process.Although microcystin-LR (MC-LR) produced by cyanobacteria has been demonstrated with strong reproductive toxicity, the mechanisms remain unclear. This study aimed to probe the effects of MC-LR on induction of autophagy in Sertoli cells, as well as the relationship between autophagy and apoptosis. After exposure to various concentrations of MC-LR for 24 or 48 h, cell viability and membrane integrity were significantly decreased under high MC-LR conditions (50–500 nM). The autophagosome marker protein LC3 was increased at mild MC-LR concentrations (0.5–5 nM). However, autophagosomes accumulated to their peak level under high MC-LR conditions in parallel with significantly up-regulated apoptosis. Treatment with an autophagy inhibitor (3-MA) abrogated autophagosome accumulation and apoptosis. This study demonstrated that MC-LR had toxic effects on Sertoli cells by inducing autophagy and apoptosis. The autophagosome accumulation may be involved in the apoptosis induced by MC-LR.

Nonylphenol (NP) is a representative endocrine disruptor that has an adverse effect on male reproduction and posses direct hazard to Sertoli cells, but the mechanism remains incompletely elucidated. In the present study, based on the structural comparability and high affinity between NP and membrane phospholipid molecules, we tested the hypothesis that entrance of NP into Sertoli cells would alter membrane biophysical characteristics and biochemical functions. First, we used gas chromatography–mass spectrometry (GC–MS) to investigate the distribution and pharmacokinetics of NP in Sertoli cells with the result revealing that NP could penetrate plasma membrane of Sertoli cells. Meanwhile, Sertoli cells treated with NP exhibited abnormal membrane potential; that is an early depolarization following short treatment and hyperpolarization after longer treatment with the highest concentration of NP. Studies on the membrane dynamics indicated that the NP exposure rendered increased membrane fluidity and decreased microviscosity and molecular order. The result of lactate dehydrogenase (LDH) leakage assay demonstrated that NP increased membrane permeability in time–dose-dependent manners. Atomic force microscopic (AFM) imaging was applied to examine the membrane topography, and the images showed that NP treatment caused disturbance of membrane topography. The activities of plasma membrane Ca2+-ATPase, Ca2+–Mg2+-ATPase and Na+–K+-ATPase were also changed following NP exposure. However, FSH receptor as an important membrane protein was not significantly altered. All the above changes led to the disturbed intracellular Ca2+ homeostasis which was an important signal triggering apoptosis. Hence, cellular membranes represented a plausible target for NP-induced cytotoxicity.

•miRNAs were altered in Sertoli cells exposed to MC-LR.•Alerted genes were involved in different cell functions including the cell morphology.•MC-LR adversely affected Sertoli cell junction formation through the regulating miRNAs.Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system.

•Microcystin-LR could enter and be accumulated in the prostate.•Perinatal MC-LR exposure directly interfered with the development of the prostate in the offspring, evidenced by prostatic necrosis, hyperplasia, inflammation, and fibrosis.•Disordered hormone conversion of androgen to estrogen may be one of the potential mechanisms of MC-LR disrupting prostate development.Although it is well known that microcystin-LR (MC-LR) may cause male reproductive toxicity, less is known on its potential impact on the development of prostate. In this study, from the 12th day in the embryonic period to the 21st day after birth, 4 randomly assigned groups of pregnant mice were exposed to 0, 1, 10, and 50 μg/L of MC-LR through drinking water followed by the analyses of their 30- and 90-day-old male offspring. The result showed that MC-LR could enter and be accumulated in the offsprings prostate. Using serological, morphological, and immunohistochemical analysis, we explored the effect of perinatal MC-LR exposure on the prostate development of male offspring. With increasing MC-LR concentrations, the 30 day-old male offspring experienced decreased prostate index, increased serum testosterone levels, decreased serum estradiol levels, and increased the serum androgen/estrogen ratio. Morphological findings showed a significant acini branching defect in both the10 and 50 μg/L group and increasing MC-LR exposures induced augmented expression of androgen receptor (AR) and estrogen receptor α (ERα). For the 90-day group, MC-LR exposure resulted in decreased physiological indexes including prostate index and the serum androgen/estrogen ratio. Pathological changes could be observed in prostate tissues of mice treated with MC-LR. Increased expression of AR and ERα was also observed. Taken together, our results demonstrated that perinatal MC-LR exposure interfered with the development of the prostate in the offspring, evidenced by prostatic necrosis, hyperplasia, inflammation, and fibrosis, anddisordered hormone conversion of androgen to estrogen inducing imbalance of androgen and estrogen in the prostate may be one of the potential mechanisms of MC-LR disrupting prostate development.