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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.

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.

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.