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

Abstract

BackgroundHeat 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.MethodsThe 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.ResultsHsp27 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).ConclusionsDownregulation 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.

Effects of Upregulation of Hsp27 Expression on Oocyte Development and Maturation Derived from Polycystic Ovary Syndrome

“…which is consistent with our previous study [15]. Interestingly, Results of the present study also showed that overexpression of Hsp27 didn't affect the rate of fertilization, and even enhanced the embryonic developmental potential of PCOS oocytes.…”

“…Our previous work demonstrated that upregulation of apoptotic-related factors Caspase 8 and Caspase 3 could increase the early apoptotic rate of oocytes, and promoted oocyte maturation in mouse oocytes. Contrastingly, the overexpression of Hsp27 in oocytes was shown to downregulate Caspase 8 and Caspase 3 expression, which depressed the early stages of apoptosis [15]. These findings were further supported by our observation that upregulated Hsp27 expression had an inhibitory effect on oocyte maturation in PCOS patients.…”

“…Interestingly, we previously found that Hsp27 was mainly expressed in human oocytes, and was downregulated in ovarian tissue isolated from women with PCOS [14]. We also found that downregulation of Hsp27 improves oocyte maturation in mice, while increasing early stage apoptosis in oocytes by inducing the activation of the extrinsic, caspase 8-mediated, apoptotic pathway [15]. Despite these important findings, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS has not been clarified.…”

Small heat shock proteins: Role in cellular functions and pathology

“…Over-expression of Hsp27 in oocytes derived from PCOS patients leads to inhibition of oocyte maturation, but improves embryonic developmental potential by down-regulating oocyte-secreted factors, BMP15 and GDF9, and the apoptoticrelated regulators, Caspase 3, 8 and 9 [353]. Downregulation of Hsp25 improved the maturation of mouse oocytes but increased early stage of apoptosis through the activation of extrinsic, caspase 8-mediated pathway [354]. Though the exact mechanism of these observations still needs to be explored, it appears that Hsp27/Hsp25 critically regulates oocyte maturation and its developmental potential through regulation of apoptosis as one of the mechanisms.…”

HSP27/HSPB1 as an adaptive podocyte antiapoptotic protein activated by high glucose and angiotensin II

“…In this sense, in oocytes HSPB1 targeting promoted caspase-3 activation in the absence of caspase-9 activation, similar to the observation in podocytes exposed to high-glucose conditions. 52 Podocyte injury results in proteinuric renal disease. 53 Contrary to MCD, both DN and FSGS are progressive glomerular diseases characterized by progressive loss of podocytes in which podocyte apoptosis has been observed.…”