| Backgroud: Oocyte development and maturation is the preparing process for fertilization and embryonic development, during which oocyte gradually gained developmental capacity. The communication with its surrounding cells is important for promoting oocyte development. Cumulus cells(CCs) can form cytoplasmic projections through the zona pellucida, establish gap junctions with plasma membrane of oocytes. Cumulus-oocyte complexes(COCs) include the oocyte and cumulus granulosa cells surrounding. It is widely believed that CCs support the development of oocytes. Most of follicles come up with atresia, which is mainly induced by granulosa cell apoptosis. It is showed that oxidative stress/reactive oxygen species(ROS) play an important role in the apoptotic mechanism. ROS damage due to aging or pathological changes significantly suppress the growth of follicle, while antioxidants can prevent the damage and apoptosis of ovarian granulosa cells. In previous studies, we constructed the protein profiles of mouse cumulus-oocyte complexes at different developmental stages. We found that the antioxidant enzymes peroxiredoxin(Prdxs) were included in those files. Prdxs are an antioxidant enzyme family that catalyze the reduction of ROS, such as H2O2 and various organic hydroperoxides to form water and alcohols. Six Prdx isoforms(Prdx1-Prdx6) have been identified in mammals. Through the mouse immature and mature COC protein profiles difference comparison, we found the expression of prdx4 was significantly up-regulated in the mature COCs(p<0.05). We thought that prdx4 should be involved in the regulation of follicular development. Previous studies indicated that Prdx4 had two isoforms, 27 kd type and 31 kd type. The 27 kd type can be secreted because of the lack of N-terminal peptide, while the 31 kd type is mainly located in cellular endoplasmic reticulum. Prdx4 knockout male mice showed testicular atrophy and oligospermia. However, little is known about the function ofprdx4 in female reproduction. This study was designed to explore the role of Prdx4 in follicular development. Using the COC in vitro cultured as a model, we observed the COC development, and detected the expressions of those factors related to oocyte development and CCs apoptosis using RNAi and Prdx4 overexpression adenoviruses. We also explored the potential mechanism of Prdx4 in either physiological or pathological conditions. These results are helpful for us to understand the follicle development, developmental disorder-related diseases and the action of Prdx4 as a new regulator, and to develop the assisted reproductive techniques of ovulation induction and immature oocytes in vitro maturing.Materials and methods Part One: Prdx4 levels in follicular fluid assosicated with IVF-ET outcomes 1. Examine the levels of Prdx4 in follicular fluid of IVF-ET patients by ELISA, and compare the Prdx4 levels between pregnant and non-pregnant patients. 2. Univariate analysis was performed using nonparametric Spearman test to explore the relationship of prdx4 with parameters of oocyte quality, such as the number of oocytes retrieved, the fertilization rate, the cleavage rate and the proportion of good quality embryos. Part Two: Effect of Prdx4 on mice follicle development 1. Examine the localization of prdx4 in human ovaries by immunohistochemistry. 2. Compare the expression of prdx4 between PCOS ovaries, premenopausal ovaries and normal ovaries by realtime PCR and western blot. 3. Compare the expression of prdx4 between human immature and mature follicle granulsa cells by realtime PCR and western blot. 4. Examine the localization of prdx4 in mice ovaries at different developmental stages by immunohistochemistry. 5. After 24 hours treatment with H2O2, expression of prdx4 in mice guanulosa cells were detected by realtime PCR and western blot. 6. After 24 hours treatment with H2O2, the guanulosa cell proliferation was tested by cell couting, MTT, Edu cell proliferation analysis, and the expression of cell cycle protein was detected by western blot.7. After 24 hours treatment with H2O2, the guanulosa cell apoptosis was analysed by flow cytometry, and the expression of apoptosis-related factor was detected by realtime PCR. Part Three: Prdx4 regulated mice follicle development 1. After H2O2 treatment in mouse COCs in vitro maturation, the morphology of the COC development was observed, including cumulus expansion and oocyte maturation. 2. After 16-18 hours of post-infection with Ad-Prdx4 or Ad-Prdx4/Si RNA adenoviruses, we validate the efficiency of Prdx4 overexpression and knockdown by realtime PCR in cumulus cells and oocytes. 3. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, the morphology of the COC development was observed and COC apoptosis was analysed by TUNEL assay. 4. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, the expressions of those apoptotic factors related to endoplasmic reticulum stress, such as CHOP10 and GRP78, were detected by realtime PCR. 5. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, c AMP levels in cumulus cells and oocytes were measured, while the oocyte maturation rate was calculated after forskolin treatment during the denuded oocytes were in vitro cultured. 6. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, the expressions of secreted fators in oocyte, such as BMP15 and GDF9, were measured by realtime PCR 7. After 16-18 hours of post-infection with Ad-Prdx4/Si RNA adenoviruses, the morphology of the COC development was observed, including cumulus expansion and oocyte maturation. 8. After 16-18 hours of post-infection with Ad-Prdx4/Si RNA adenoviruses and treatment with different concentrations of H2O2, the morphology of the COC development was observed.Results Part One: Prdx4 levels in follicular fluid assosicated with IVF-ET outcomes 1. Prdx4 levels in follicle fluid of pregnant patients were significantly higher than those in non-pregnant patients(PCOS group: 2.0±1.61 ng/ml vs. 17.09±1.26 ng/ml, p<0.05; control group: 22.26±1.65 ng/ml vs. 16.43±0.97 ng/ml, p<0.01). 2. Prdx4 levels in follicle fluid were significantly and positively correlated to the oocyte fertilization rates(r=0.334; p=0.011) and the good quality embryo rates(r=0.326; p=0.013). Part Two: Effect of Prdx4 on mice follicle development 1. Prdx4 was mainly located in granulosa cells of human ovaries. 2. Expression of Prdx4 in ovarian tissues of patients with polycystic ovary syndrome and premenopausal ovaries were significantly lower than that in control, at both m RNA and protein levels(p<0.05). 3. Expression of prdx4 in the matured follicle granulosa cells was twice higher than that in immature granulosa cells, at both m RNA and protein levels(p<0.05). 4. Prdx4 was predominantly localized in mice granulosa cells and the expressison pattern showed the highest level in mature stage, while lower level in adolescence and aging stages(p<0.05). 5. After 24 hours treatment with H2O2 in mice guanulosa cells, expressions of Prdx4 m RNA and protein were increased in a concentration-dependent manner, with the highest level when 200 μM of H2O2 treatment(p<0.05). When H2O2 concentration reached to 400 μM or higher, prdx4 expression decreased rapidly. 6. Cell counting, MTT and Ed U incorporation assays all showed a concentration-dependent decrease in guanulosa cell proliferation following exposure to H2O2 treatment. The significant inhibition was observed since 400 μM H2O2 treatment(p<0.05). Low concentrations of H2O2 up-regulated expressions of CDK1, CDK4, p21, and p15. 7. After 24 hours treatment with H2O2, guanulosa cell apoptosis showed a concentration-dependent increase. The apoptosis rate reached to 35% when exposed to 400 μM H2O2(p<0.05), with a significantly increased expressions of thoseapoptosis-related factors at m RNA level, such as caspase 3, caspase 9, caspase 12 and Bcl-2(p<0.01). Part Three: Prdx4 regulated mice follicle development 1. After the mice COC in vitro maturation was treated with H2O2, the cumulus expansion index and the oocyte maturation rate showed concentration-dependent decrease(p<0.05). 2. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses, expression of prdx4 m RNA in cumulus cells other than oocyte was triple increased when compared with the Ad-GFP infected group(p<0.05). After 16-18 hours of post-infection with Ad-Prdx4/Si RNA adenoviruses, expression of prdx4 m RNA in cumulus cells was more than 60% decreased when compared with the Ad-GFP infected group(p<0.05). 3. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, the cumulus expansion index and the oocyte maturation rate significant increased when compare with H2O2 treatment group(p<0.05), while the COC apoptosis rate were reduced after prdx4 over-expressed and Tiron treatment(p<0.05). 4. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, expressions of those endoplasmic reticulum stress related factors, such as CHOP10 and GRP78, were significantly decreased(p<0.05). 5. Cumulus cells and oocyte were mechanically separated before 16-18 hours of infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, the oocyte maturation rate showed no difference between groups(p>0.05). 6. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, c AMP levels in both cumulus cells and oocytes increased, while both significantly decreased in the Ad-Prdx4 and Tiron treatment groups(p<0.05). After Forskolin were added into the medium of denuded oocytes culture, the oocyte maturation rate was similar with the H2O2 treatment only group(p>0.05). 7. After 16-18 hours of post-infection with Ad-Prdx4 adenoviruses or Tiron treatment combined with H2O2, expressions of oocyte-secreted fators, such as GDF9and BMP15, were significantly increased, while significantly decreased in the Ad-Prdx4 and Tiron treatment groups(p<0.05). 8. After 16-18 hours of post-infection with Ad-Prdx4/Si RNA adenoviruses, the morphology of COC development showed no significant change when compared with the Ad-GFP/Si RNA group(p>0.05). 9. After 16-18 hours of post-infection with Ad-Prdx4/Si RNA adenoviruses combined with different concentrations of H2O2, the cumulus expansion index and the oocyte maturation rate showed concentration-dependent decreases.Conclusion 1. Prdx4 levels in follicle fluid were strongly associated with the clinical pregnancy outcome. Prdx4 levels were significantly correlated with oocyte fertilization and the good quality embryo rates(r=0.334; p=0.011 and r=0.326; p=0.013, respectively), which suggested that Prdx4 was related with the oocyte quality and the following pregnancy outcomes due to its regulation of intra-follicular oxidative damage. 2. Prdx4 protein was predominantly localized in ovarian granulosa cells. Prdx4 expression significantly altered during ovarian development and aging, which suggested that the Prdx4 expressed in granulosa cells participated in the regulation of follicle development and maturation. 3. The potential mechanisms of Prdx4 include: the Prdx4 expressed in follicular granulosa cells acts as an antioxidant to regulate the cell apoptosis in follicular development. Prdx4 also regulates oocyte development by c AMP via gap junctions. 4. Our study indicated firstly that Prdx4 in ovaries participated in regulation of follicular development and maturation by regulating the cellular oxidative stress. The decreased expression of Prdx4 in PCOS and premenopausal ovaries indicates a potential pathological mechanism of ovulation disorders and potential target for clinical further study. |
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