| BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women of reproductive age and a clinical pathological model to study oocyte development. PCOS is the most common cause of female infertility, affecting5to10%of women during their reproductive age. It is characterized by anovulatory infertility, hyperandrogenism, insulin resistance and paracine dysregulation, all of which could perturb the intra-follicular environment. Since the PCOS ovary exhibits abnormal apoptotic activity and folliculogenesis, it is a clinical pathological model for studying oocyte maturation and development. Understanding how PCOS is related to abnormalities in extra-and intra-ovarian factors and their impact on granulosa cell (GC)-oocyte interactions, oocyte maturation and potential embryonic developmental competence, is crucial to improving fertility and optimizing controlled ovarian hyperstimulation, thus enhancing pregnancy outcomes in women with PCOS that undertake IVF treatment. Clinical management of women with PCOS undergoing IVF treatment has been well-described. Although PCOS patients are typically characterized by producing an increased number of oocytes after controlled ovarian hyperstimulation, they are often of poor quality leading to lower rates of fertilization, cleavage and implantation, and a higher miscarriage rate. Hence, other factors, aside from chromosomal factors, are most likely associated with the significantly increased risk of pregnancy loss in patients with PCOS. Impaired oocyte maturation and embryonic developmental competence in PCOS women is possibly linked with abnormal endocrine/paracrine factors, metabolic dysfunction and alterations in the intrafollicular microenvironment during folliculogenesis and follicle maturation.Currently, little is known about the cause and pathophysiological mechanism of PCOS. However, a lot of researchers presented that an apoptosis and anti-apoptosis imbalance occurs in PCOS ovaries. Heat shock protein27(Hsp27), a member of the small heat shock protein family, is an apoptotic regulator which inhibits apoptosis via multiple pathways. As a molecular chaperone protein, Hsp27is involved in cellular protection in response to a variety of stresses such as heat shock, toxicants, injury and oxidative stress. Emerging evidence showed that Hsp27had strong anti-apoptotic properties by interacting directly with the caspase activation components in apoptotic pathways, consequently exerting protective effects in apoptosis-related injuries. Hsp27has been shown to protect cells against apoptosis by binding with cytochrome c, inhibiting the activation of caspase9and blocking the extrinsic Fas-and TNF-mediated apoptotic pathways. Interestingly, we found that Hsp27was mainly expressed in the human oocyte, and was down-regulated in the ovaries of women with PCOS in our previous study. Also we found that down-regulation of Hsp27improved the maturation of oocytes in mouse, while it increased the early stage of apoptosis in oocytes by inducing the activation of the extrinsic, caspase8-mediated pathway.Many studies have focused on heat shock proteins as regulators of cell death and survival. Although the exact mechanism of the role of Hsp in anti-apoptosis is unclear, evidence demonstrates that Hsp has an important function during fertilization and embryo development process. There are few reports about the effects of Hsp27on oocyte maturation and development competence. We hypothesized that in the PCOS ovary, apoptotic activity is disrupted, and follicle development and maturation is blocked. Our previous study confirmed that Hsp27can regulate oocyte maturation, whilst Hsp27expression is lower in PCOS patients which could cause disruption of apotosis. This might result in an increase in the number of small follicles which do not go through atresia normally and so cannot generate dominant follicles. However, the relationship between this apparent problem with oocyte development and low expression of Hsp27is unclear. PCOS patients undergoing IVF treatment obtain a large number of oocytes after stimulation with gonadotropins but embryo development potential and implantation rate are lower than normal. As it is unclear whether these observations are associated with low expression of Hsp27, we attempted to investigate the exact effect of Hsp27on oocyte maturation and developmental competence in PCOS. In addition, we wanted to discuss what role apoptosis plays during oocyte maturation and embryo development.Methods and Results1. Hsp27expression and location in the development of mouse preimplantation embryo.Fertilized oocytes were collected from6-week-old female ICR mice and cultured in vitro in microdrops covered by mineral oil. After culture for24h,48h,72h and96h, embryos were collected at the different developmental stages. To determine the expression and location of Hsp27during embryo development, real time RT-PCR, Western blot, and immunofluorescence was performed on the preimplantion embryos at different development stages. Our results showed that Hsp27expression was mainly in the cytoplasm and nuclei (except the nucleolus) of mouse embryos. Immunofluorescence showed that Hsp27expression dramatically increased during embryo development.2. The role of Hsp27in the mouse preimplantation embryo development.The activity of Hsp27was blocked at the protein level through microinjection of Hsp27antibody. Our results showed the formation rate of blastocysts (84.8%) was not significantly different to that in two controls (81.80%,83.1%). Total cell number of blastocysts was not different betweenthe three groups. AdCMV-Hsp27was microinjected into the cytoplasm of zygotes at the two pronuclei stage to improve the activity of Hsp27. The formation rate of blastocystss (84.5%) was also not significantly different from those of two controls (83.8%,86.4%, P>0.05). Expression of Hsp27at the protein level was detected by immunofluorescence at the8-cell stage. The blastocyst formation rate of mouse zygotes was not significantly different after microinjection of AdSiRNA-Hsp27when compared with negative controls (83.1%vs86.3%and87.7%, P>0.05). Expression of Hsp27at protein level was detected by immunofluorescence at8-cell stage.3. Effects of Upregulation of Hsp27Expression on Oocyte Development and Maturation of the PCOS Oocytes3.1Morphological observationAs GFP gene was contained in the up-regulated vectors, the expression level of Hsp27in injected oocytes can be evaluated by the brightness of green fluorescence observed under a fluorescence microscope. The results showed that the maturation rate of pAdTrack-CMV-hHsp27-injected oocytes was significantly lower than those of control (33.5%vs55.9%and61.4%)(P<0.05),3.2Expression of oocyte-specific secreted factors by RT-PCR Our results showed that mRNA expression of Bmp15(definition) and Gdf9(definition) decreased when Hsp27expression increased (P<0.05), which was in accordance with morphological observations and suggested a close relation between Hsp27and oocyte maturation.3.3Impediment of Hsp27up-regulated to expression of apoptotic-related regulatorsResults showed that the expression level of Caspase8,、 Caspase9and Caspase3in oocytes in the pAdTrack-CMV-hHsp27-injected group were significantly lower than that of the pAdTrack-CMV-injected group (P<0.05) while the expression of Cyt c showed no significant difference, suggesting that Hsp27down-regulation may activate the Caspase8-mediated apoptotic pathway and also affect the intrinsic Caspase9-mediated pathway.4. Fertilization and embryonic development potential after Hsp27up-regulated.The effect of experimental over-expression of Hsp27in oocytes from PCOS patients on fertilization rate and embryonic developmental potential was assessed. Our results showed that the fertilization rate and the high quality embryo rate at day3were not significantly different in oocytes expressing elevated levels of Hsp27versus the control. However, the blastocyst formation rate was significant higher than controls (41.30%versus23.53%).Conclusion1. Hsp27expression was recognized mainly in the cytoplasm and nuclei (except the nucleolus) of mouse embryos. Immunofluorescence showed that Hsp27expression dramatically increased during embryo development. Hsp27regulated oocyte maturation by early apoptosis but did not affect pre-implantation embryonic development as a single factor.2. In PCOS patients, Hsp27is involved in the maturation of oocytes by regulating apoptosis. Hsp27regulated the maturation and apoptosis in oocytes by inducing the activation of an extrinsic, caspase8-mediated pathway.3. Our results demonstrated that up-regulation of Hsp27expression could inhibit maturation of oocytes from PCOS patients, while improving embryonic development potential. Further studies are required to elucidate how Hsp27affects oocyte maturation and development potential. |
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