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This study used response surface methodology to determine the optimal conditions for extraction of polysaccharides from Pyracantha. fortuneana (PSPF), and studied the mechanism of PSPF-inducing apoptosis in human ovarian carcinoma Skov3 cells. Response surface methodology (RSM) were adopted to extract PSPF. The maximum value of polysaccharide yield was obtained under these optimal conditions. PSPF had good potential as an antioxidant. Exposure of cells to PSPF resulted in cytotoxicity through the induction of apoptosis, and the reactive oxygen species were increased, mitochondrial membrane potential decreased, DNA damage (detected as γ- H2AX and RAD51 foci) was observed in Skov3 cells. In addition, PSPF could induce apoptosis of cancer cells. Therefore, PSPF should be explored as novel potential antioxidants and an anti-tumor drug in a clinical setting.

Di(2-ethylhexyl)phthalate (DEHP) has proven characteristics of an endocrine-disrupting compound (EDC), which can threaten the reproductive health of humans and other animals. In mammals, a series of chromosomal events occur during the meiotic stage of oocytes. External toxins may enter the body and cause infertility and other related diseases. Therefore, it is crucial to explore the influence of DEHP exposure on the molecular mechanism of germ cell meiosis. We used single-cell RNA sequencing (scRNA-seq) to analyse the ovaries of foetal mice at embryonic day 12.5 (E12.5) and E14.5 after maternal DEHP exposure. DEHP exposure further activated the pathways related to DNA repair in germ cells, increased the expression of genes related to DNA damage and changed the developmental trajectory of germ cells. DEHP exposure may affect the proliferation of pregranulosa (PG) cells. Moreover, DEHP exposure altered the signal transduction between PG cells and germ cells. We showed that DEHP affects meiosis by causing DNA damage in oocytes and disrupting the signal transduction between PG cells and germ cells. These results provide a strong theoretical basis for the prevention and treatment of DEHP-mediated female reproductive health problems.

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer which is widely used in the manufacture of plastics. As a common environmental contaminant and recognized endocrine disrupting chemical, DEHP is able to deregulate the functions of a variety of tissues, including the reproductive system both in males and females. In order to investigate the possible effects of DEHP on the first wave of folliculogenesis, occurring in the mouse ovary postnatally, mice were administered 20 or 40 μg/kg DEHP through intraperitoneal injection at days 5, 10 and 15 post partum (dpp). Following DEHP treatment the gene expression profile of control and exposed ovaries was compared by microarray analyses at 20 dpp. We found that in the exposed ovaries DEHP significantly altered the transcript levels of several immune response and steroidogenesis associated genes. In particular, DEHP significantly decreased the expression of genes essential for androgen synthesis by theca cells including Lhcgr, Cyp17a1, Star and Ldlr. Immunohistochemistry and immune flow cytometry confirmed reduced expression of LHCGR and CYP17A1 proteins in the exposed theca cells. These effects were associated to a significant reduction in ovarian concentrations of progesterone, 17β-estradiol and androstenedione along with a reduction of LH in the serum. Although we did not find a significant reduction of the number of primary, secondary or antral follicles in the DEHP exposed ovaries when compared to controls, we did observe that theca cells showed an altered structure of the nuclear envelope, fewer mitochondria, and mitochondria with a reduced number of cristae. Collectively, these results demonstrate a deleterious effect of DEHP exposure on ovarian steroidogenesis during the first wave of folliculogenesis that could potentially affect the correct establishment of the hypothalamic-pituitary-ovarian axis and the onset of puberty.

Zearalenone (ZEA), a metabolite of Fusarium fungi, is commonly found on moldy grains. Because it can competitively combine to estrogen receptor to disrupt estrogenic signaling, it has been reported to have serious adverse effects on animal reproduction systems. In order to explore the genotoxic effects of ZEA exposure on ovarian somatic cells, porcine granulosa cells were exposed to 10 μM and 30 μM ZEA for 24 or 72 h in vitro. The results showed that ZEA exposure for 24 h remarkably reduced the proliferation of porcine granulosa cells in a dose-dependent manner as determined by MTT analysis and flow cytometry. Furthermore, exposure to ZEA for 72 h induced apoptosis, and RNA sequence analysis also revealed that the expression of apoptosis related genes were altered. RT-qPCR, immunofluorescence and western blot analysis further confirmed the expression of DNA damage and repair related genes (γ-H2AX, BRCA1, RAD51 and PRKDC) were increased in ZEA exposed granulosa cells. When the estrogen antagonist, tamoxifen, was added with ZEA in the culture medium, the DNA damage and repairment by ZEA returned to normal level. Collectively, these results illustrate that ZEA disrupts genome stability and inhibits growth of porcine granulosa cells via the estrogen receptors which may promote granulosa cell apoptosis when the DNA repair system is not enough to rescue this serious damage.

Ding-Ping Bai,1,* Xi-Feng Zhang,2,* Guo-Liang Zhang,3,4 Yi-Fan Huang,1 Sangiliyandi Gurunathan5 1Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, China; 2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China; 3Dong-E-E-Jiao Co., Ltd., Shandong, China; 4National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Shandong, China; 5Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea *These authors contributed equally to this work Background: Zinc oxide nanoparticles (ZnO NPs) are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3). Methods: ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. Results: Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H2AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy. Finally, the Western blot analysis revealed upregulation of Bax, caspase-9, Rad51, γ-H2AX, p53, and LC3 and downregulation of Bcl-2. Conclusion: The study findings demonstrated that the ZnO NPs are able to induce significant cytotoxicity, apoptosis, and autophagy in human ovarian cells through reactive oxygen species generation and oxidative stress. Therefore, this study suggests that ZnO NPs are suitable and inherent anticancer agents due to their several favorable characteristic features including favorable band gap, electrostatic charge, surface chemistry, and potentiation of redox cycling cascades. Keywords: zinc oxide nanoparticles, human ovarian cancer cells SKOV3, mitochondrial membrane potential, apoptosis, DNA fragmentation, autophagy

Xi-Feng Zhang,1,2 Feng-Hua Huang,1 Guo-Liang Zhang,3 Ding-Ping Bai,4 De Felici Massimo,5 Yi-Fan Huang,4 Sangiliyandi Gurunathan6 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China; 2Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao, China; 3National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Dong-E-E-Jiao Co., Ltd, DongE, Shandong, China; 4Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, China; 5Department of Biomedicine and Prevention, University of Rome ‘Tor Vergata’, Rome, Italy; 6Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea Background: Recently, there has been much interest in the field of nanomedicine to improve prevention, diagnosis, and treatment. Combination therapy seems to be most effective when two different molecules that work by different mechanisms are combined at low dose, thereby decreasing the possibility of drug resistance and occurrence of unbearable side effects. Based on this consideration, the study was designed to investigate the combination effect of reduced graphene oxide-silver nanoparticles (rGO-AgNPs) and trichostatin A (TSA) in human ovarian cancer cells (SKOV3). Methods: The rGO-AgNPs were synthesized using a biomolecule called lycopene, and the resultant product was characterized by various analytical techniques. The combination effect of rGO-Ag and TSA was investigated in SKOV3 cells using various cellular assays such as cell viability, cytotoxicity, and immunofluorescence analysis. Results: AgNPs were uniformly distributed on the surface of graphene sheet with an average size between 10 and 50 nm. rGO-Ag and TSA were found to inhibit cell viability in a dose-dependent manner. The combination of rGO-Ag and TSA at low concentration showed a significant effect on cell viability, and increased cytotoxicity by increasing the level of malondialdehyde and decreasing the level of glutathione, and also causing mitochondrial dysfunction. Furthermore, the combination of rGO-Ag and TSA had a more pronounced effect on DNA fragmentation and double-strand breaks, and eventually induced apoptosis. Conclusion: This study is the first to report that the combination of rGO-Ag and TSA can cause potential cytotoxicity and also induce significantly greater cell death compared to either rGO-Ag alone or TSA alone in SKOV3 cells by various mechanisms including reactive oxygen species generation, mitochondrial dysfunction, and DNA damage. Therefore, this combination chemotherapy could be possibly used in advanced cancers that are not suitable for radiation therapy or surgical treatment and facilitate overcoming tumor resistance and disease progression. Keywords: graphene, trichostatin, cytotoxicity, reactive oxygen species, apoptosis, DNA fragmentation, double-strand DNA breaks

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling. The objective of this study was to investigate the expression of Grb7 and its clinicopathological significance in cervical cancer. Utilising immunohistochemical staining, we examined the expression of Grb7 in 120 cases of human cervical cancer tissue and 10 cases of adjacent non-cancerous cervical tissue. The positive rate of Grb7 protein expression was 34.2%, which was significantly higher than that in adjacent non-cancerous cervical tissue (0%, p

A retrospective study of consecutive patients. The purpose of this study was to compare the clinical effect of biomimetic mineralized collagen (MC) modified polymethylmethacrylate (PMMA) bone cement and traditional PMMA bone cement for the treatment of osteoporotic vertebral compression fractures (OVCF). New fracture on adjacent level is the major postoperative complication of percutaneous vertebroplasty (PVP). The clinical incidence was 12.4% to 27.7%. The increased stiffness of the treated vertebral body caused by filling bone cement is considered as one of the main reasons. A total of 30 patients treated with traditional PMMA bone cement from June 2013 to March 2016 were selected as the traditional group, while 50 patients treated with MC modified PMMA bone cement from July 2014 to March 2016 were selected as the modified group. The 2 groups were compared by injection time of the bone cements, postoperative pain relief effects, vertebral height restoration, CT value changes of the treated vertebral bodies, and postoperative complications in the clinical observations. The surgeries were successfully completed in both groups. In the treatment of OVCF, the MC modified bone cement was able to achieve the same pain relief and vertebral height restoration effects compared to traditional bone cement during the follow-ups, although the injection time of the cement was prolonged in the operation. MC modified bone cement significantly reduced the incidence of postoperative adjacent vertebral fracture from 13.3% to 2%, and significantly increased bone density of the treated vertebral bodies. The MC modified PMMA bone cement showed good clinical outcomes and better mechanical properties than the traditional bone cements.

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.

Xi-Feng Zhang,1 Sangiliyandi Gurunathan2 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul, South Korea Abstract: Ovarian cancer is one of the most important malignancies, and the origin, detection, and pathogenesis of epithelial ovarian cancer remain elusive. Although many cancer drugs have been developed to dramatically reduce the size of tumors, most cancers eventually relapse, posing a critical problem to overcome. Hence, it is necessary to identify possible alternative therapeutic approaches to reduce the mortality rate of this devastating disease. To identify alternative approaches, we first synthesized silver nanoparticles (AgNPs) using a novel bacterium called Bacillus clausii. The synthesized AgNPs were homogenous and spherical in shape, with an average size of 16–20nm, which are known to cause cytotoxicity in various types of human cancer cells, whereas salinomycin (Sal) is able to kill cancer stem cells. Therefore, we selected both Sal and AgNPs to study their combined effect on apoptosis and autophagy in ovarian cancer cells. The cells treated with either Sal or AgNPs showed a dose-dependent effect with inhibitory concentration (IC)-50 values of 6.0µM and 8µg/mL for Sal and AgNPs, respectively. To determine the combination effect, we measured the IC25 values of both Sal and AgNPs (3.0µM and 4µg/mL), which showed a more dramatic inhibitory effect on cell viability and cell morphology than either Sal or AgNPs alone. The combination of Sal and AgNPs had more pronounced effect on cytotoxicity and expression of apoptotic genes and also significantly induced the accumulation of autophagolysosomes, which was associated with mitochondrial dysfunction and loss of cell viability. Our data show a strong synergistic interaction between Sal and AgNPs in tested cancer cells. The combination treatment increased the therapeutic potential and demonstrated the relevant targeted therapy for the treatment of ovarian cancer. Furthermore, we provide, for the first time, a mode of action for Sal and AgNPs in ovarian cancer cells: enhanced apoptosis and autophagy. Keywords: apoptosis, autophagy, cell viability, caspase activity, ovarian cancer, salinomycin, silver nanoparticles

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

To evaluate the clinical effects of CT-guided percutaneous puncture and local chemotherapy for lumbosacral spinal tuberculosis.From January 2002 to March 2013, 145 patients (84 males and 61 females) with lumbosacral spinal tuberculosis underwent failed conservative treatment were treated with CT-guided minimally invasive surgery. Their clinical data were retrospectively analyzed. Among them, 143 patients were followed up, aged from 2.5 to 81 years with an average of (42.60 +/- 17.14) years. Fourteen cases (6 cases with internal fixation and 8 cases without internal fixation) recurred and 32 cases complicated with paraspinal abscess after operation. Preoperatively 1 case complicated with lower limb weakness and superficial sensation worse, 1 case with muscle strength decrease, this 2 cases were grade D of Frankle, other cases were grade E. And 1 patient underwent fenestration operation and local chemotherapy, 144 cases with CT-guided percutaneous puncture and local chemotherapy. ESR and lumbar lordosis angle of all patients were observed preoperatively and final follow-up.The mean follow-up time was 67 months (ranged, 21 to 149 months) in 143 cases, and 73 cases more than 5 years. All patients obtained clinical healing. ESR was (44.96 +/- 12.41) mm/h before operation and (7.25 +/- 3.43) mm/h at final follow-up, there was significant difference between preoperative and postoperative (t=35.06, P=0.000). Lumbar lordosis angle was (36.32 +/- 8.55) degrees before operation and (33.35 +/- 8.16) at final follow-up, there was significant difference between preoperative and postoperative (t=13.90, P=0.000).When conservative treatment fails for 3 months or more, the patients have good spinal stabilization, nerve function is more than grade D of Frankel, CT-guided percutaneous puncture and local chemotherapy can get satisfactory outcomes for lumbosacral spinal tuberculosis.

Primordial follicle formation and the subsequent transition of follicles through primary and secondary stages constitute crucial events of oogenesis. In particular, in mammals, defects in the processes that precede and accompany the formation of the primordial follicle pool can affect the size of this population significantly, while alterations in follicle activation, growth and maturation can result in premature depletion of the follicle reserve or cause follicle arrest at immature stages. Over the last decade, in vitro and in vivo approaches have been used to provide evidence that exposure to di(2-ethylhexyl)phthalate(DEHP), the most widely used plasticizer, has a deleterious effect on various stages of folliculogenesis in rodents. There is growing concern, supported by epidemiological and experimental data, that DEHP may have similar effects in women. This article reviews the evidence, with particular reference to our own findings, that DEHP may actually exert a variety of adverse effects on mammalian folliculogenesis from early to final stages of oogenesis, including altered development of the primordial germ cells, impaired fetal oocyte survival and meiotic progression, reduced oocyte nest breakdown, acceleration of primordial follicle activation, altered follicle steroidogenesis and increased follicle atresia. These effects can cause serious complications for reproductive and nonreproductive women's health. In addition, emerging data indicate that phthalates, including DEHP, may cause subtle epigenetic changes in germ cells that can be transmitted to subsequent generations, with potential negative effects on human health. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc.

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 μg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.

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.

The widely used diethylhexyl phthalate (DEHP) is a known endocrine disruptor that causes persistent alterations in the structure and function of female reproductive system, including ovaries, uterus and oviducts. To explore the molecular mechanism of the effect of DEHP on the development of mammary glands, we investigated the cell cycle, growth, proliferation and gene expression of mammary gland cells of pregnant mice exposed to DEHP. It was demonstrated, for the first time, that the mammary gland cells of pregnant mice treated with DEHP for 0.5–3.5 days post-coitum had increased proliferation, growth rate and number of cells in the G2/S phase. The expression of cell proliferation-related genes was significantly altered after short time and low-dose DEHP treatment of mammary gland cells in vivo and in vitro. These findings showed adverse effects of DEHP on mammary gland cells in pregnant mice.

Oxidative stress (OS), as a signal of aberrant intracellular mechanisms, plays key roles in maintaining homeostasis for organisms. The occurrence of OS due to the disorder of normal cellular redox balance indicates the overproduction of reactive oxygen species (ROS) and/or deficiency of antioxidants. Once the balance is broken down, repression of oxidative stress is one of the most effective ways to alleviate it. Ongoing studies provide remarkable evidence that oxidative stress is involved in reproductive toxicity induced by various stimuli, such as environmental toxicants and food toxicity. Zearalenone (ZEA), as a toxic compound existing in contaminated food products, is found to induce mycotoxicosis that has a significant impact on the reproduction of domestic animals, especially pigs. However, there is no information about how ROS and oxidative stress is involved in the influence of ZEA on porcine granulosa cells, or whether the stress can be rescued by curcumin. In this study, ZEA-induced effect on porcine granulosa cells was investigated at low concentrations (15 μM, 30 μM and 60 μM). In vitro ROS levels, the mRNA level and activity of superoxide dismutase, glutathione peroxidase and catalase were obtained. The results showed that in comparison with negative control, ZEA increased oxidative stress with higher ROS levels, reduced the expression and activity of antioxidative enzymes, increased the intensity of fluorogenic probes 2’, 7’-Dichlorodihydrofluorescin diacetate and dihydroethidium in flow cytometry assay and fluorescence microscopy. Meanwhile, the activity of glutathione (GSH) did not change obviously following 60 μM ZEA treatment. Furthermore, the underlying protective mechanisms of curcumin on the ZEA-treated porcine granulosa cells were investigated. The data revealed that curcumin pre-treatment significantly suppressed ZEA-induced oxidative stress. Collectively, porcine granulosa cells were sensitive to ZEA, which may induce oxidative stress. The findings from this study clearly demonstrate that curcumin is effective to reduce the dysregulation of cellular redox balance on porcine granulosa cells in vitro and should be further investigated for its protective role against ZEA in animals.

Activins can exert several roles in ovary development. However, little is known about their involvement in early mammalian oogenesis. In this study, we reported that activin receptors (including ActRIA, ActRIB, ActRIIA, and ActRIIB) are expressed throughout the development of the mouse ovaries from 12.5 days postcoitum (dpc) to 21 days postparturition (dpp). Moreover, we found that in vitro, the addition of activin A (ActA) to the culture medium of 12.5 dpc ovarian tissues accelerated the progression of oocytes throughout meiotic prophase I stages. This result was reproduced in vivo following administration of ActA to pregnant mice. The in vitro effect of ActA was associated with increased expression of premeiotic and meiotic genes (including Dazl, Spo11, Stra8, Scp3, and Rec8) in the ovarian tissues. Mechanistically, ActA-dependent SMAD3 signaling modulated the expression of members of the retinoic acid (RA) system, including the RA degradation CYP26B1 enzyme and the RA receptors. Finally, ActA promoted the survival and growth of fetal and early postnatal oocytes and primordial follicle assembly both in vitro and in vivo. In conclusion, the present study identifies new roles of ActA in early oogenesis and suggested that ActA and RA might cooperate in promoting meiosis in female germ cells.

The Notch and transforming growth factor (TGF)-β 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-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β 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-β 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-β 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.

Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 μM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development.

The female reproductive lifespan is largely determined by the size of primordial follicle pool, which is established following germ cell cyst breakdown around birth. Almost two-third of oocytes are lost during germ cell cysts breakdown, following autophagic and apoptosis mechanisms. To investigate a possible relationship between germ cell cyst breakdown and nutrition supply, we established a starvation model in mouse pups at birth and evaluated the dynamics of cyst breakdown during nutrient deprivation. Our results showed that after 36 h of starvation between 1.5 and 3 d.p.p., indicators of metabolism both at systemic and ovarian level were significantly altered and the germ cell cyst breakdown markedly decreased. We also found that markers of oxidative stress, autophagy and apoptosis were increased and higher number of oocytes in cyst showing autophagic markers and of TUNEL-positive oocytes and somatic cells were present in the ovaries of starved pups. Moreover, the proliferation of pre-granulosa cells and the expression of the oocyte-specific transcription factor Nobox were decreased in such ovaries. Finally, we observed that the ovaries of the starved pups could recover a normal number of follicles after about 3 weeks from re-feeding. In conclusion, these data indicate that nutrient deficiency at birth can generate a number of adaptive metabolic and oxidative responses in the ovaries causing increased apoptosis both in the somatic cells and oocyte and autophagy mainly in these latter and leading to a delay of germ cell cyst breakdown and follicle assembly.

Bisphenol-A (BPA) and diethylhexyl phthalate (DEHP) are estrogenic compounds widely used in commercial plastic products. Previous studies have shown that exposure to such compounds have adverse effects on various aspects of mammalian reproduction including folliculogenesis. The objective of this study was to examine the effects of BPA and DEHP exposure on primordial follicle formation. We found that germ cell nest breakdown and primordial follicle assembly were significantly reduced when newborn mouse ovaries were exposed to 10 or 100 μM BPA and DEHP in vitro. Moreover, BPA and DEHP exposure increased the number of TUNEL positive oocytes and the mRNA level of the pro-apoptotic gene Bax in oocytes. These effects were associated with decreased expression of oocyte specific genes such as LIM homeobox 8 (Lhx8), factor in the germline alpha (Figla), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and newborn ovary homeobox (Nobox). Interestingly, BPA and DEHP exposure also prevented DNA demethylation of CpG sites of the Lhx8 gene in oocytes, a process normally associated with folliculogenesis. Finally, folliculogenesis was severely impaired in BPA and DEHP exposed ovaries after transplantation into the kidney capsules of immunodeficient mice. In conclusion, BPA and DEHP exposures impair mouse primordial follicle assembly in vitro.

To investigate the feasibility of minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) using hybrid internal fixation of pedicle screws and a translaminar facet screw for recurrent lumbar disc herniation.From January 2010 to December 2011, 16 recurrent lumbar disc herniation patients, 10 male and 6 female patients with an average age of 45 years (35-68 years) were treated with unilateral incision MIS-TLIF through working channel. After decompression, interbody fusion and fixation using unilateral pedicle screws, a translaminar facet screw was inserted from the same incision through spinous process and laminar to the other side facet joint. The results of perioperative parameters, radiographic images and clinical outcomes were assessed. The repeated measure analysis of variance was applied in the scores of visual analogue scale (VAS) and Oswestry disablity index (ODI).All patients MIS-TLIF were accomplished under working channel including decompression, interbody fusion and hybrid fixation without any neural complication. The average operative time was (148 ± 75) minutes, the average operative blood loss was (186 ± 226) ml, the average postoperative ambulation time was (32 ± 15) hours, and the average hospitalization time was (6 ± 4) days. The average length of incision was (29 ± 4) mm, and the average length of translaminar facets screw was (52 ± 6) mm. The mean follow-up was 16.5 months with a range of 12-24 months. The postoperative X-ray and CT images showed good position of the hybrid internal fixation, and all facets screws penetrate through facets joint. The significant improvement could be found in back pain VAS, leg pain VAS and ODI scores between preoperative 1 day and postoperative follow-up at all time-points (back pain VAS:F = 52.845, P = 0.000;leg pain VAS:F = 113.480, P = 0.000;ODI:F = 36.665, P = 0.000).Recurrent lumbar disc herniation could be treated with MIS-TLIF using hybrid fixation through unilateral incision, and the advantage including less invasion and quickly recovery.

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.

To investigate the feasibility and safety of unilateral incision hybrid fixation using pedicle screws and a translaminar screw in minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF).From January to June 2010, 18 patients with single-level lumbar disc disease were treated with MIS-TLIF under METRx(TM) X-tube. After decompression and fixation using unilateral pedicle screws, a translaminar screw was inserted from the same incision to the other side. The results of perioperative parameters, radiographic images and clinical outcomes were assessed.All patients underwent MIS-TLIF were accomplished unilateral hybrid fixation without any neural complication. The average operative time was (107 ± 19) min, the average operative blood loss was (62 ± 21) ml, and the average postoperative ambulation time was (21 ± 5) h. The average length of translaminar facets screw was (52 ± 2) mm, and the postoperative images showed all screws penetrate through facets joint. During the follow-up the visual analogue scale and Oswestry disability index scores were significant improved compared with preoperative (F = 42.221 - 259.833, P0.01).Bilateral hybrid fixation could be completed through unilateral incision by pedicle screws and a translaminar screw in MIS-TLIF, and the advantage including less invasion, quickly recovery, short operative time, and saving fixation cost.

To summarize and discuss the lapsus and the treatment of the lumbar intervertebral disc herniation using percutaneous endoscopic lumbar discectomy (PELD).Between July 2002 and October 2010, 689 patients with lumbar intervertebral disc herniation treated by PELD were analyzed, including 448 males, and 241 females. Single lumbar intervertebral disc herniation were 669 cases. double lumbar intervertebral disc herniation were 19; three lumbar intervertebral disc herniation were 1. Central type in 66, side central type in 365, lateral type in 242, extreme lateral type in 10, sequestered type in 6. These cases with complications in operation and postoperation were studied retrospectively.There were nucleus pulposus omissions in 5 patients and 2 patients underwent open resection of nucleus pulposus during operation immediately and the second operation was needed in 3 cases, 1 case with transforaminal lumbar interbody fusion (TLIF) and the others with open resection of nucleus pulposus. Two patients had nerve root injury, but all completely recovered in 3 - 6 months after operation. Spinal dura mater disruption was in 2 patients, recovered after suturing of skin wound. All 689 patients were followed up for 6 - 96 months, mean follow-up time was 33 months. Postoperative spondylodiscitis was in 7 patients, recovery after expectant treatment in 1, percutaneous puncture irrigation and drainage for continued use of local antibiotics in 4, posterior infective lumbar discectomy in 2. Postoperative relapse was in 6 patients, operated secondly by PELD in 4 and by TLIF in 2, recovery after the second operation. Nerve root induced hyperalgesia and burning-like nerve root pain was seen in 19 patients, the symptom was improved by analgesic drug, neurotrophy drug and physiotherapy. The effect of single segment PELD was not good in 10 patients with spinal stenosis, who underwent multiple segment TLIF later.The complications during operation usually are nucleus pulposus omissions, nerve root injury, spinal dura mater disruption. Accordingly the complications after operation include spondylodiscitis, recurrence, nerve root induced hyperalgesia or burning-like nerve root pain. Strict indication, aseptic technique, skilled operation and proper rehabilitation exercise are effective ways to reduce complications.

Xi-Feng Zhang,1,2 Jung-Hyun Park,1 Yun-Jung Choi,1 Min-Hee Kang,1 Sangiliyandi Gurunathan,1 Jin-Hoi Kim11Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea; 2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of ChinaBackground: Silver nanoparticles (AgNPs) have attracted much interest and have been used for antibacterial, antifungal, anticancer, and antiangiogenic applications because of their unique properties. The increased usage of AgNPs leads to a potential hazard to human health. However, the potential effects of AgNPs on animal models are not clear. This study was designed to investigate the potential impact of AgNPs on pregnant mice.Methods: The synthesis of AgNPs was performed using culture extracts of Bacillus cereus. The synthesized AgNPs were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy. AgNPs were administrated into pregnant mice via intravenous infusion at 1.0 mg/kg doses at 6.5 days postcoitum (dpc). At 13.5, 15.5, and 17.5 dpc, the pregnant mice were euthanized, and the embryo and placenta were isolated. The meiotic status of oocytes was evaluated. DNA methylation studies were performed, and aberrant imprinting disrupted fetal, placental, and postnatal development. Quantitative real-time polymerase chain reaction analysis and Western blot were used to analyze various gene expressions.Results: The synthesized AgNPs were uniformly distributed and were spherical in shape with an average size of 8 nm. AgNPs exposure increased the meiotic progression of female germ cells in the fetal mouse ovaries, and maternal AgNP exposure significantly disrupted imprinted gene expression in 15.5 dpc embryos and placentas, such as Ascl2, Snrpn, Kcnq1ot1, Peg3, Zac1, H19, Igf2r, and Igf2; DNA methylation studies revealed that AgNPs exposure significantly altered the methylation levels of differentially methylated regions of Zac1.Conclusion: The results from this study indicated that early exposure to AgNPs has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta. These results can contribute to research involved in the safe use of various biomedical applications of AgNPs and improves the understanding of the development of AgNPs in biomedical applications.Keywords: silver nanoparticles, methylation, meiosis, gene expression

Bisphenol A (BPA), a synthetic additive used to harden polycarbonate plastics and epoxy resin, is ubiquitous in our everyday environment. Many studies have indicated detrimental effects of BPA on the mammalian reproductive abilities. This study is aimed to test the potential effects of BPA on methylation of imprinted genes during oocyte growth and meiotic maturation in CD-1 mice. Our results demonstrated that BPA exposure resulted in hypomethylation of imprinted gene Igf2r and Peg3 during oocyte growth, and enhanced estrogen receptor (ER) expression at the levels of mRNA and protein. The relationship between ER expression and imprinted gene hypomethylation was substantiated using an ER inhibitor, ICI182780. In addition, BPA promoted the primordial to primary follicle transition, thereby speeding up the depletion of the primordial follicle pool, and suppressed the meiotic maturation of oocytes because of abnormal spindle assembling in meiosis I. In conclusion, neonatal exposure to BPA inhibits methylation of imprinted genes during oogenesis via the ER signaling pathway in CD-1 mice.

Recently, we reported the identification of a previously uncharacterized and evolutionarily conserved gene, fragile-site associated tumor suppressor (FATS), at a frequently deleted region in irradiation (IR)-induced tumors. However, the role of FATS in breast cancer development and its clinical significance has not been defined. The aim of this study was to determine the role of FATS in breast cancer development and to evaluate its clinical significance in breast cancer.The expression level of FATS mRNA was determined in 106 breast carcinomas and 23 paired normal breast tissues using quantitative real time reverse transcription-polymerase chain reaction (RT-PCR). The relationship between FATS expression and clinicopathological parameters were also analyzed.The mRNA level of FATS was down-regulated in breast cancer compared with paired normal tissues. Low expression of FATS was correlated with high nuclear grade. There was a tendency to a favorable outcome for patients with high expression of FATS (P = 0.346). However, low expression of FATS was associated with poor outcome of breast cancer patients with node positive (P = 0.011). Furthermore, the mRNA level of FATS showed an independent value in predicting the outcome of breast cancer patients with positive lymph nodes.FATS is involved in the carcinogenesis and development of breast cancer and could be a potential biomarker and prognostic factor for breast cancer therapy.

Diethylhexyl phthalate (DEHP) is a widely used industrial additive for increasing plastic flexibility. It disrupts the physiological functions of endogenous hormones and induces abnormal development of mammals. The objectives of the present study were to evaluate the effects of DEHP exposure on ovarian development of pregnant mice and whether the effects are inheritable. We found that the synthesis of oestradiol in pregnant mice after DEHP exposure was significantly decreased, and that the first meiotic progression of female fetal germ cells was delayed. Furthermore, the DNA methylation level of Stra8 was increased and the expression levels of Stra8 were significantly decreased. An accelerated rate of follicle recruitment in F1 mice was responsible for the depletion of the primordial-follicle pool. Maternal DEHP exposure also significantly accelerated the recruitment of primordial follicles in F2 mice. In conclusion, our results indicated that maternal DEHP exposure induced ovarian development deficiency, which was transgenerational in mice.

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.