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Zearalenone (ZEN), one of the most prevalent non-steroidal oestrogenic mycotoxins, is primarily produced by Fusarium fungi. Due to its toxicity as an oestrogenic compound and wide distribution in feed and foods, the reproductive toxicology of ZEN exposure is of public concern. The aim of the present study was to investigate the effect of ZEN on Sertoli cells to identify apoptotic pathways induced by this compound. We found that ZEN reduced the viability and caused apoptosis in Sertoli cells in vitro. Notably, we observed that such effects were associated with a significant increase in reactive oxygen species (ROS) and the number of cells that showed positive staining for γH2AX and RAD51, enzymes essential for repairing DNA damage. There was a parallel decrease in the expression of occludin and connexin 43, proteins that are present in the testis-blood barrier and gap junctions of Sertoli cells, respectively. Overall, the present study confirms that ZEN exposure can have serious deleterious effects on mammalian Sertoli cells and offers novel insight about its molecular targets in these cells.

In this study, the synthesis parameters of the lotus root polysaccharide iron complex (LRPF) were determined and optimized by response surface methodology. Under the optimum preparation conditions, the pH of the solution was 9, the ratio of M (trisodium citrate): m (lotus root polysaccharide) was 0.45, the reaction time was 3 h. UV spectroscopy, thermogravimetry, FT-IR spectroscopy, X-ray diffraction, CD, and NMR were used for the characterization of the LRPF. LRPF has good stability and easily releases iron ions under artificial gastrointestinal conditions. LRPF exhibited antioxidant activity in vitro and can significantly improve the antioxidant activity in vivo. In addition, LRPF has a good effect in the treatment of iron deficiency anemia in model mice, impacts the gut microbiome, and reduces the iron deficiency-induced perniciousness by regulating steroid hormone biosynthesis. Therefore, LRPF can be used as a nutritional supplement to treat and prevent iron-deficiency anemia and improve human immunity.

Considering that research has mainly focussed on how excessive iron supplementation leads to reproductive cytotoxicity, there is a lack of in-depth research on reproductive system disorders caused by iron deficiency. To gain a better understanding of the effects of iron deficiency on the reproductive system, especially spermatogenesis, we first constructed a mouse model of iron deficiency. We employed multi-omic analysis, including transcriptomics, metabolomics, and microbiomics, to comprehensively dissect the impact of iron deficiency on spermatogenesis. Moreover, we verified our findings in detail using western blot, immunofluorescence, immunohistochemistry, qRT-PCR and other techniques. Microbiomic analysis revealed altered gut microbiota in iron-deficient mice, and functional predictive analysis showed that gut microbiota can regulate spermatogenesis. The transcriptomic data indicated that iron deficiency directly alters expression of meiosis-related genes. Transcriptome data also revealed that iron deficiency indirectly regulates spermatogenesis by affecting hormone synthesis, findings confirmed by metabolomic data, western blot and immunofluorescence. Interestingly, competing endogenous RNA networks also play a vital role in regulating spermatogenesis after iron deficiency. Taken together, the data elucidate that iron deficiency impairs spermatogenesis and increases the risk of male infertility by affecting hormone synthesis and promoting gut microbiota imbalance.

Acute gouty arthritis (AGA) is characterized by the accumulation of pro‑inflammatory cytokines, which are immunological responses to monosodium urate (MSU) crystals. It has been demonstrated that long non‑coding RNA (lncRNA)‑MM2P is a novel regulator of M2 polarization of macrophages. The aim of the present study was to investigate whether lncRNA‑MM2P regulates the MSU‑induced inflammatory process. In cell models of RAW 264.7 and THP‑1‑derived macrophages, decreased expression of lncRNA‑MM2P was observed in lipopolysaccharide‑ and MSU‑treated macrophages, which was accompanied with obvious inflammatory responses. Using small interfering RNA to knockdown lncRNA‑MM2P led to the upregulation of MSU‑mediated inflammatory responses, both in RAW 264.7 and THP‑1‑derived macrophages. In conclusion, lncRNA‑MM2P could be an important regulator of MSU‑induced inflammation, and therefore could be involved in the development of AGA.

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.

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 Biotechnology, Konkuk University, Seoul, Republic of Korea Abstract: Graphene has been shown much interest, both in academics and industry due to its extraordinary physical, chemical, and biological proprieties. It shows great promises in biotechnological and biomedical applications as an antibacterial and anticancer agent, nanocarrier, sensor, etc. However, many studies demonstrated the toxicity of graphene in several cell lines, which is an obstacle to its use in biomedical applications. In this study, to improve the biocompatibility of graphene, we used nicotinamide (NAM) as a reducing and stabilizing agent to catalyze the reduction of graphene oxide (GO) to reduced graphene oxide (rGO). The resulted smaller-sized GO (NAM-rGO) showed excellent biocompatibility with mouse embryonic fibroblast cells, evidenced by various cellular assays. Furthermore, NAM-rGO had no effect on mitochondrial membrane permeability and caspase-3 activity compared to GO. Reverse transcription polymerase chain reaction analysis allowed us to identify the molecular mechanisms responsible for NAM-rGO-induced biocompatibility. NAM-rGO significantly induced the expression of genes encoding tight junction proteins (TJPs) such as zona occludens-1 (Tjp1) and claudins (Cldn3) without any effect on the expression of cytoskeleton proteins. Furthermore, NAM-rGO enhances the expression of alkaline phosphatase (ALP) gene, and it does this in a time-dependent manner. Overall, our study depicted the molecular mechanisms underlying NAM-rGO biocompatibility depending on upregulation of TJPs and ALP. This potential quality of graphene could be used in diverse applications including tissue regeneration and tissue engineering. Keywords: biocompatibility, graphene oxide, nicotinamide, reduced graphene oxide, tight junction proteins, alkaline phosphatase

Zhe Han,1,* Qi Yan,1,* Wei Ge,2 Zhi-Guo Liu,1 Sangiliyandi Gurunathan,3 Massimo De Felici,4 Wei Shen,2 Xi-Feng Zhang1 1College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China; 2Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, People’s Republic of China; 3Department of Stem Cell and Regenerative Biology, Konkuk University, Seoul, Republic of Korea; 4Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy *These authors contributed equally tothis work Background: Nanoscience and nanotechnology are developing rapidly, and the applications of nanoparticles (NPs) have been found in several fields. At present, NPs are widely used in traditional consumer and industrial products, however, the properties and safety of NPs are still unclear and there are concerns about their potential environmental and health effects. The aim of the present study was to investigate the potential toxicity of ZnO NPs on testicular cells using both in vitro and in vivo systems in a mouse experimental model. Methods: ZnO NPs with a crystalline size of 70 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy, and atomic force microscopy. The cytotoxicity of the ZnO NPs was examined in vitro on Leydig cell and Sertoli cell lines, and in vivo on the testes of CD1 mice injected with single doses of ZnO NPs.Results: ZnO NPs were internalized by Leydig cells and Sertoli cells, and this resulted in cytotoxicity in a time- and dose-dependent manner through the induction of apoptosis. Apoptosis likely occurred as a consequence of DNA damage (detected as γ-H2AX and RAD51 foci) caused by increase in reactive oxygen species associated with loss of mitochondrial membrane potential. In addition, injection of ZnO NPs in male mice caused structural alterations in the seminiferous epithelium and sperm abnormalities.Conclusion: These results demonstrate that ZnO NPs have the potential to induce apoptosis in testicular cells likely through DNA damage caused by reactive oxygen species, with possible adverse consequences for spermatogenesis and therefore, male fertility. This suggests that evaluating the potential impacts of engineered NPs is essential prior to their mass production, to address both the environmental and human health concerns and also to develop sustainable and safer nanomaterials. Keywords: ZnO nanoparticle, Sertoli cells, Leydig cells, mice

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.

The present study aims to investigate the protective effects of ω-3 polyunsaturated fatty acids (ω-3PUFAs) against high-fat diet induced male mouse reproductive dysfunction and to explore circadian regulation mechanisms. Male C57BL/6 mice were randomly divided into three groups and fed a normal chow diet (control group, CON), a high-fat diet (HFD group) or a HFD supplemented with fish oil (FO group) for 12 weeks. After 12 weeks of feeding, the body weight and the ratio of perinephric and epididymal fat weight to body weight were significantly higher in the HFD group compared with the CON group. The supplement of fish oil rich in ω-3PUFAs only slightly reduced the HFD-induced obesity but remarkably ameliorated HFD-induced dyslipidemia, sexual hormones disorder, testicle lesions and germ cell apoptosis. Fish oil supplementation restored the expression of steroid synthesis associated genes in HFD fed mouse and flattened the HFD-induced oscillations in circadian genes' expression. Fish oil supplementation prevented HFD-induced male mouse reproductive dysfunction and modified the rhythmic expression of testosterone synthesis related genes.

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.

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.

Silver nanoparticles (AgNPs) have attracted increased interest and are currently used in various industries including medicine, cosmetics, textiles, electronics, and pharmaceuticals, owing to their unique physical and chemical properties, particularly as antimicrobial and anticancer agents. Recently, several studies have reported both beneficial and toxic effects of AgNPs on various prokaryotic and eukaryotic systems. To develop nanoparticles for mediated therapy, several laboratories have used a variety of cell lines under in vitro conditions to evaluate the properties, mode of action, differential responses, and mechanisms of action of AgNPs. In vitro models are simple, cost-effective, rapid, and can be used to easily assess efficacy and performance. The cytotoxicity, genotoxicity, and biocompatibility of AgNPs depend on many factors such as size, shape, surface charge, surface coating, solubility, concentration, surface functionalization, distribution of particles, mode of entry, mode of action, growth media, exposure time, and cell type. Cellular responses to AgNPs are different in each cell type and depend on the physical and chemical nature of AgNPs. This review evaluates significant contributions to the literature on biological applications of AgNPs. It begins with an introduction to AgNPs, with particular attention to their overall impact on cellular effects. The main objective of this review is to elucidate the reasons for different cell types exhibiting differential responses to nanoparticles even when they possess similar size, shape, and other parameters. Firstly, we discuss the cellular effects of AgNPs on a variety of cell lines; Secondly, we discuss the mechanisms of action of AgNPs in various cellular systems, and try to elucidate how AgNPs interact with different mammalian cell lines and produce significant effects; Finally, we discuss the cellular activation of various signaling molecules in response to AgNPs, and conclude with future perspectives on research into AgNPs.

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.

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.

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.

Xi-Feng Zhang,* Yun-Jung Choi,* Jae Woong Han, Eunsu Kim, Jung Hyun Park, Sangiliyandi Gurunathan, Jin-Hoi Kim Department of Animal Biotechnology, Konkuk University, Seoul, South Korea *These authors contributed equally tothis work Background: Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs). Methods: Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results: AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs. Conclusion: Our results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required. Keywords: Sertoli cells, Leydig cells, apoptosis, oxidative stress, tight junction proteins, apoptosis

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 evaluate the effect of a 10-day course of triptolide (TP) on rat cytochrome (CY) P3A4 activity, and on the pharmacokinetics of cyclophosphamide (CPA).In the pharmacokinetics experiment, rats were orally given 0.9% NaCl solution (n=5) and TP [1.2 (mg/kg·d)] for 10 days and a single dose of CPA was administered intravenously (100 mg/kg) to rats on day 11. Blood samples were collected up to 4 h at predetermined time intervals, the plasma concentration of CPA was determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were determined. In the in vitro CYP3A4 activity inhibition research, rat blank liver microsomes were divided into 3 groups: a control group, a TS (5 μL, 200 μmol/L) with TP (5 μL, 12.5 μmol/L) group, a TS with ketoconazole (5 μL, 1 μmol/L) group. Concentration of 6β-hydroxylated testosterone (6β-OHT) in liver microsomes was measured by HPLC and the activity of CYP 3A4 was calculated through the following formula: Einhibitor/Econtrol × 100%=Cinhibitor/Ccontrol × 100%.Compared with the control group, the area under the plasma concentration-time curve (AUC0-∞) of CPA was significantly increased by 229.05% pretreated with TP (P0.01). Peak plasma concentrations (Cmax) of CPA was significantly increased and plasma half-life was correspondingly extended. The CYP3A4 activity was significantly inhibited by ketoconazole 93.5%±0.2% and TP 84.6%±0.3% compared with the control group (P0.01 and P0.05, respectively).Our results strongly suggested that long-term oral intake of TP can distinctly inhibit the CYP3A4 activity and this inhibition evidently decrease the formation of toxic metabolites of CPA.

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.

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

Xi-Feng Zhang,1,2 Sangiliyandi Gurunathan,1 Jin-Hoi Kim1 1Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea; 2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, People’s Republic of China Background: Metal nanoparticles (MNPs) play an important role in consumer products. An increasing use of MNPs has raised concerns about potential risks for human health. Therefore, in vivo tests of MNPs are urgently required. Using mice as a model animal, the aim of the present study was designed to investigate the effect of biologically synthesized silver nanoparticles (AgNPs) on spermatogenesis in neonatal mice. Methods: AgNPs were synthesized using Bacillus funiculus. The prepared nanoparticles were characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, and transmission electron microscopy. The prepared AgNPs were used to investigate testis development in neonatal mice. Institute of Cancer Research neonatal male mice were used in all experiments and were treated with different doses (0, 1, and 5 mg/kg) of AgNPs five times (interval of 3 days from postnatal day [PND] 8–21) by abdominal subcutaneous injection. Results: The results showed that the sperm abnormalities such as quality and quantity were significantly increased by the synthesized AgNPs. The diameter of the convoluted tubules shrank significantly in mice treated with AgNPs on PND28 and PND42. The results of reverse transcription-quantitative polymerase chain reaction indicated that the E1f1ay, Gsta4, and Fdx1 genes were up-regulated, and the Amh, Cx43, and Claudin-11 genes were down-regulated in response to AgNPs exposure on PND28; however, these genes recovered at PND60. AgNPs had no effect on the recombination levels of chromosomes in germ cells. Conclusion: These results demonstrated the adverse effects of AgNPs on the male reproductive tract, particularly spermatogenesis and the quality of sperm. This study suggests that the development of nanomaterials should be safer and non-toxic to the living organisms and the potential reprotoxicity of AgNPs should be investigated more carefully. Keywords: silver nanoparticles, male mouse, sperm, gene expression, testis development

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.

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.

Genetic structure and character of Chaidamu goats were studied through simple random sampling. Genetic structure was analysed from five aspects, and phylogeny status was also investigated. The results indicated that: (1) the average phenotypic heterogeneity degree of coat color and morphological character were 0.3419 and 0.5207, respectively; (2) polymorphous blood albumen existed in 6 loci and the average loci heterozygosity was 0.2584; and (3) polymorphism existed in marked genes by DND-RAPD with diversity of 0.4085 approximately 0.5318. Phylogeny status was investigated through clustering by Ward's method on Chaidamu Goats and other domestic goats. All these indicated that Chaidamu Goats was a domestic goat with less intensively selected breed.

Bisphenol A (BPA), a chemical used in many consumer products, interferes with the endocrine system of mammals, including humans. The aim of the present study was to investigate the effect of BPA on spermatogenesis and semen quality. The objective of this study was to assess the effects of BPA on mouse spermatogenesis. CD1 mice were used in all experiments. Mice were treated with different doses of BPA (0, 20 and 40 μg kg–1 day–1from postnatal Day (PND) 3 to PND21, PND 35 or PND49. After 5 weeks BPA treatment, oestrogen receptor α expression was increased in mouse testis, whereas the meiotic progression of germ cells was slowed. Thus, both the quality and quantity of spermatozoa were decreased in 7-week-old mice. However, BPA had no effect on DNA methylation of imprinted genes such as Igf2, Igf2r, Peg3 and H19, in germ cells. In addition, exposure of male mice to BPA resulted in abnormal offspring that were smaller with a low-quality pelage when they were 35 days old. In conclusion, BPA hampers spermatogenesis and the subsequent development of offspring.

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.