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Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

ABSTRACT: BACKGROUND: Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified. METHODS: The expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot. RESULTS: Hsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9). CONCLUSIONS: Downregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.

SUBMITTER: Liu JJ

PROVIDER: S-EPMC2890611 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

Liu Jin-Juan JJ Ma Xiang X Cai Ling-Bo LB Cui Yu-Gui YG Liu Jia-Yin JY

Reproductive biology and endocrinology : RB&E 20100514

<h4>Background</h4>Heat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not ye ...[more]

PMID: 20465849

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Project description:Study questionDoes exposure to bisphenol-A (BPA) affect the maturation of human oocytes in vitro?Summary answerThere was a dose-response association of BPA exposure with altered human oocyte maturation in vitro.What is known alreadyThere is widespread exposure of the general population to BPA. BPA has been detected in the human follicular fluid. Animal studies have shown that BPA exposure is associated with maturation arrest and spindle abnormalities in maturing oocytes.Study design, size, durationA randomized trial, using 352 clinically discarded oocytes from 121 patients.Participants/materials, setting, methodsThe study population was drawn from patients undergoing IVF/ICSI cycles in our program at Brigham and Women's Hospital from March 2011 to April 2012. Oocytes from only one cycle for each patient were included in the study. Cycles with at least two germinal vesicle stage oocytes were included with random allocation of one oocyte to culture for 30 h without BPA and remaining sibling oocytes to medium-containing BPA (20, 200 ng/ml or 20 µg/ml). Oocytes were fixed and labeled for tubulin, actin and chromatin and examined with immunofluorescence and confocal microscopy. Oocytes were assessed for meiotic stage (n = 292), and those at metaphase II (MII, n = 175) were further classified according to their spindle configurations and patterns of chromosome alignment. McNemar's test was used to compare dichotomized maturation status. Generalized estimating equations were used to account for the correlation between oocytes from the same woman and for the spindle analysis.Main results and the role of chanceAs the BPA dose increased, there was a decrease in the percentage of oocytes that progressed to MII (P = 0.002) and increases in the percentage of oocytes that were degenerated (P = 0.01) or that had undergone spontaneous activation (P = 0.007). Among MII oocytes, as the BPA dose increased, there was a significant trend (by test for trend) for a decreased incidence of bipolar spindles (P < 0.0001) and aligned chromosomes (P = 0.02).Limitations, reasons for cautionAlthough we used sibling oocytes to overcome potential confounders, such as infertility diagnosis and maternal age, additional studies with a larger number of oocytes are required to confirm the present results. Having access only to clinically discarded oocytes, we were limited to evaluating only those oocytes that failed to mature in vivo despite having been exposed to gonadotrophin stimulation and the ovulatory trigger of HCG.Wider implications of the findingsTo our knowledge, this is the first study investigating the effect of BPA on oocyte meiotic maturation, spindle morphology and chromosome alignment in human oocytes. Together with prior animal studies, the data support the negative influences of BPA on cell cycle progression, spindle architecture and chromosome organization during oocyte maturation. Furthermore, the increased rates of abnormal maturation in oocytes exposed to BPA may be relevant to our understanding of the decrease in fertility reported in the last decades.Study funding/competing interest(s)This study was funded by the NIEHS Center Grant Pilot Project (P30-ES000002). R.M. was sponsored by a fellowship from the Environmental Health Fund, Israel and by the Frederick L. Hisaw Endowment, Harvard School of Public Health. There are no conflicts of interest.Trial registration numbern/a.

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Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

Publications

Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro.

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