The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In germinal vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the germinal vesicle. Shortly after germinal vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The germinal vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after germinal vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I–telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in germinal vesicle breakdown and the anaphase–telophase transition.
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Li-Jun Huo, Cheng-Guang Liang, Ling-Zhu Yu, Zhi-Sheng Zhong, Zeng-Ming Yang, Heng-Yu Fan, Da-Yuan Chen, and Qing-Yuan Sun
Qiuling Jie, Lijun Chen, Jiangying Liang, Xiaohui Yang, Fei Sun, and Yanlin Ma
In brief
Preeclampsia is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes, but the mechanisms underlying the development of preeclampsia are not fully understood. This study shows that ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9 and is involved in the pathogenesis of preeclampsia.
Abstract
Preeclampsia (PE) is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes. However, the mechanisms underlying the development of PE are not fully understood. ETS Variant Transcription Factor 4 (ETV4) plays an important role in cell proliferation, migration, and invasion. In this study, we aimed to explore the potential function of ETV4 in placental trophoblast cells. We analyzed the expression and location of ETV4 in PE and uncomplicated placental tissues using RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence staining. The results showed that both the mRNA and protein levels of ETV4 were markedly decreased in PE placental tissues compared with placental tissues from women with uncomplicated pregnancies (P < 0.05). Then, the effects of ETV4 on HTR-8/SVneo and Bewo cell proliferation, migration, and invasion were evaluated by MTT, 5-ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays, respectively. The results showed that ETV4 knockdown inhibited both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). Conversely, overexpression of ETV4 promoted both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). We then measured the expression of MMP-2 and MMP-9 in HTR8/SVneo cells. We found that ETV4 knockdown decreased the mRNA and protein expression of MMP-2 and MMP-9, while ETV4 overexpression increased MMP-2 and MMP-9 mRNA and protein expression (P < 0.05). In conclusion, ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9. Our findings provide novel insight into the mechanisms underlying the occurrence of PE.