Yanni Feng

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

Diethylhexyl phthalate (DEHP) is an estrogen‐like compound widely used as a commercial plasticizer and present in medical devices, tubing, food containers and packaging. It is considered an endocrine disruptor and studies on experimental animals showed that exposure to DEHP can alter the function of several organs including liver, kidneys, lungs and reproductive system, particularly the developing testes of prenatal and neonatal males. Exposure to DEHP has been proposed as a potential human health hazard. This study assessed the effects of DEHP on folliculogenesis and oocyte maturation using the mouse as the experimental model. Newborn female mice were hypodermically injected with DEHP at doses of 20 and 40 μg/kg per body weight following different exposure regimens during the weaning period. We found that DEHP altered both folliculogenesis and oocyte development. In particular, DEHP exposure significantly decreased the number of the primordial follicles at pubertal and adult age by possibly accelerating the rate of follicle recruitment dynamics, reduced and/or delayed the level of imprinted gene methylation in the oocytes and increased metaphase II spindle abnormalities in oocytes matured in vitro. Furthermore, the weight of pups and litter size of mothers exposed to DEHP were significantly lower than controls. Finally, the number of primordial follicles appeared significantly reduced also in the F1 offspring at the adult age. These results show that DEHP may have a number of adverse effects on oogenesis, especially when exposure occurs during early postnatal age, arising concerns about the exposure of human female infants and children to this compound. Environ. Mol. Mutagen. 54:354–361, 2013.

Recovery of Functional Oocytes from Cultured Premeiotic Germ Cells After Kidney Capsule Transplantation

The efficiency of in vitro culture systems for a premeiotic female germ cell is still low, mostly because of our incomplete understanding of the mechanisms controlling oogenesis and the obvious difficulties in reproducing the complex in vivo environment of such a process under in vitro conditions. Here we explored the possibility of recovering the developmental potential of mouse oocytes generated in vitro from premeiotic germ cells by transplantation under a kidney capsule of adult animals. To this aim, mouse embryonic ovaries of 12.5 days postcoitum cultured in vitro in a serum-free medium for 7 or 14 days, were transplanted beneath the kidney capsule of immunodeficient mice and analyzed after 21 (7+21 group) or 14 days (14+14 group). Cultured ovaries before transplantation showed delayed oocyte meiotic progression and follicle development. Interestingly, grafted ovaries of both groups, especially those of the 7+21 group, seemed able to restore the reproductive cycle of recipients. While the almost complete absence of primordial follicles was observed in grafted ovaries, oocytes from these ovaries showed transcript levels of genes associated to oocyte maturation similar to control. Moreover, the developmental stage of follicles and oocytes of the 7+21 group ovaries were comparable to that of 21 days post partum in vivo ovaries, whereas significant developmental delay were found in the 14+14 group ovaries. Nevertheless, oocytes retrieved from transplanted ovaries of both groups matured (around 80%) and were fertilized in vitro (around 20%-45%). Two-cell embryos from the fertilized oocytes developed to hatching blastocysts (about 50%) or gave rise to healthy live offspring (from 6% to 10%) when transplanted in a host mother. In conclusion, our results indicate that premeiotic female germ cells cultured in vitro up to primordial/primary follicle stages preserve their capability to complete oogenesis and can be fertilized and generate live pups after transplantation into a suitable in vivo environment.

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

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

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

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

Zinc Oxide Nanoparticles Influence Microflora in Ileal Digesta and Correlate Well with Blood Metabolites

Zinc oxide nanoparticles (ZnO NPs) are used widely in consumer and industrial products, however, their influence on gut microbiota and metabolism and their mutual interactions are not fully understood. In this study, the effects of ZnO NPs on ileal bacterial communities, plasma metabolites, and correlations between them were investigated. Hens were fed with different concentrations of ZnO NPs [based on Zn; 0 mg/kg (control), 25 mg/kg, 50 mg/kg, and 100 mg/kg] for 9 weeks. Subsequently, ileal digesta and blood plasma were collected for analysis of microflora and metabolites, respectively. The V3-V4 region of the 16S rRNA gene of ileal digesta microbiota was sequenced using the Illumina HiSeq 2500 platform. The predominant bacterial community in the ileum belongs to the phylum Firmicutes. The richness of the bacterial community was negatively correlated with increasing amounts of ZnO NPs (r = -0.636, P < 0.01); when ZnO NP levels were at 100 mg/kg, microbiota diversity was significantly decreased (P < 0.05). The community structure determined by LEfSe analysis indicated that Bacilli, Fusobacteria, and Proteobacteria were changed, and Lactobacillus was reduced by ZnO NPs. Moreover, metabolism as analyzed by nuclear magnetic resonance (NMR) indicated that glucose, some amino acids, and other metabolites were changed by ZnO NPs. Choline, lactate, and methionine were positively correlated with bacterial richness. In summary, ZnO NPs could influence the levels of microflora in ileal digesta, particularly Lactobacillus. Furthermore, the richness of the microbiota was related to changes in choline, lactate, and methionine metabolism.

Regulation of steroid hormones and energy status with cysteamine and its effect on spermatogenesis

Although it is well known that cysteamine is a potent chemical for treating many diseases including cystinosis and it has many adverse effects, the effect of cysteamine on spermatogenesis is as yet unknown. Therefore the objective of this investigation was to explore the effects of cysteamine on spermatogenesis and the underlying mechanisms. Sheep were treated with vehicle control, 10mg/kg or 20mg/kg cysteamine for six months. After that, the semen samples were collected to determine the spermatozoa motility by computer-assisted sperm assay method. Blood samples were collected to detect the levels of hormones and the activity of enzymes. Spermatozoa and testis samples were collected to study the mechanism of cysteamines actions. It was found that the effects of cysteamine on spermatogenesis were dose dependent. A low dose (10mg/kg) cysteamine treatment increased ovine spermatozoa motility; however, a higher dose (20mg/kg) decreased both spermatozoa concentration and motility. This decrease might be due to a reduction in steroid hormone production by the testis, a reduction in energy in the testis and spermatozoa, a disruption in the blood-testis barrier, or a breakdown in the vital signaling pathways involved in spermatogenesis. The inhibitory effects of cysteamine on sheep spermatogenesis may be used to model its effects on young male patients with cystinosis or other diseases that are treated with this drug. Further studies on spermatogenesis that focus on patients treated with cysteamine during the peripubertal stage are warranted.

MicroRNA-126 participates in lipid metabolism in mammary epithelial cells

Lipids are a major component of milk and are important for infant growth and development. MicroRNA-126 (miR-126) has previously been observed in mammary glands and adipocytes and is known to be involved in lipid metabolism during the process of atherosclerosis. However, it remains unknown whether miR-126 also participates in lipid metabolism in mammary luminal epithelial cells (MECs). In the current investigation, miR-126-3p inhibition stimulated lipid synthesis in MECs in part through increasing levels of the lipid synthesis enzymes FASN, ACSL1, and Insig1. Overexpression of miR-126-3p decreased lipid content in MECs with a reduction in FASN and Insig1. Furthermore, the expression of miR-126-3p was diminished by the steroid hormones estradiol and progesterone with a subsequent elevation of lipid formation in MECs. We also noted that miR-126-3p was expressed differentially at various stages of murine mammary gland development, exhibiting a negative correlation with FASN. Together these findings suggest that miR-126-3 might be involved in lipid metabolism in mammary gland.

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

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

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

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.

MicroRNA-221 may be involved in lipid metabolism in mammary epithelial cells

Milk lipids, important for infant growth and development, are produced and secreted by mammary gland under the regulation of steroid hormones, growth factors, and microRNAs (miRNAs). miR-221 has been identified in milk and adipocytes and it plays important roles in regulating normal mammary epithelial hierarchy and breast cancer stem cells; however, its roles in lipid metabolism in mammary epithelial cells (MECs), the cells of lipid synthesis and secretion, are as yet unknown. Through overexpression or inhibition of miR-221 expression, we found that it regulated lipid metabolism in MECs and was expressed differentially at various stages during murine mammary gland development. Inhibition of miR-221 expression increased lipid content in MECs through elevation of the lipid synthesis enzyme FASN, while overexpression of miR-221 reduced MEC lipid content. Moreover, the steroid hormones estradiol and progesterone decreased miR-221 expression with a subsequent increase in lipid formation in MECs. The expression of miR-221 was lower during lactation, which suggests that it may be involved in milk production. Therefore, miR-221 might be a useful target for influencing milk lipid production.