The Study On Changes Of Gene And Protein Expression And Activity Of CTSB, CTSS And Caspase 3 In Sheep Oocytes

Cathespin B(CTSB) and cathespin S(CTSS) are two sorts of cysteine protease in lysosomal, playing an important role in cell apoptosis, maturation of oocytes and implantation of embryos. Caspase 3 is the executor in apoptosis, and the activation of caspase 3 is the marker of early stage of apoptosis. Studies on changes of expression and activity of these three kinds of protease in oocytes during in vitro maturation(IVM) and correlations with quality and developmental competence of oocytes, and effects of E-64, cysteine inhibitor, on CTSB, CTSS and caspase 3, are able to detect the apoptosis stimulation to oocytes and take theory support for improving in vitro developmental competence of oocytes.RT-PCR was utilized to detect gene expression of CTSB, CTSS and caspase 3 oocytes from different sources and with different quality. Protein expression and activity of CTSB, CTSS and caspase 3 in oocytes were detected with double sandwich ELSIA.Comparied with oocytes with poor quality, CTSB、CTSS and caspase 3 gene and protein expression and activity were significantly or very significantly higher in oocytes with normal quality(P<0.05;P<0.01). Compared with lamb oocytes, CTSB, CTSS and caspases 3 gene and protein expression and CTSB activity in adult sheep oocytes were significantly or very significantly lower in adult sheep oocytes(P<0.05;P<0.01).Results showed that CTSB m RNA abundance kept stable in adult sheep oocytes, 1μmol/L E-64 and 5μmol/L E-64 had no significant effects on it, and 10μmol/L E-64 group was very significantly higher in CTSB m RNA abundance compared with control group; CTSS m RNA was hardly to be detected before IVM 16 h,and there was no significant differences in CTSS transcripts between groups; caspase 3 m RNA abundance fluctuated in the process of IVM(P<0.05). 1μmol/L E-64 had no significant effects on it(P>0.05), 5μmol/L E-64 reduced caspase 3 m RNA abundance(P<0.05), and 10μmol/L E-64 up-regulated caspase 3 gene expression(P<0.05). CTSB m RNA increased in lamb oocytes(P<0.01), 1μmol/L E-64 and 5μmol/L E-64 embarassed the improvement of CTSB m RNA abundance(P<0.05;P<0.01), 10μmol/L E-64 very significantly promoted CTSB gene expression(P<0.01); what was different from adult sheep oocytes, CTSS m RNA was capable to be detected in lamb ooctes at IVM 0h, and it significantly increased in control group at IVM 24h(P<0.05). 1μmol/L E-64 had no significant effects on CTSS gene expression(P>0.05), 5μmol/L E-64 decreased CTSS m RNA abundance(P<0.05), and 10μmol/L E-64 very significantly promoted CTSS gene expression(P<0.01), changes of caspase 3 m RNA abundance, and the difference is CTSS transcript in 1μmol/L E-64 and 5μmol/L E-64 groups had no significant difference with the one at IVM 0h(P>0.05).CTSB、CTSS and caspase 3 protein abundance and activity were detected with ELISA method. In adult sheep oocytes, CTSB activity and protein abundance kept stable(P>0.05). 5μmol/L E-64 merely decrease CTSB activity at IVM 8h(P<0.05), while had no significant effects on protein abundance(P>0.05). 10μmol/L E-64 significantly inhibited CTSB activity(P<0.01)and promoted protein abundance(P<0.05).CTSS activity and protein abundance did not change significantly(P>0.05), 5μmol/L E-64 and 10μmol/L E-64 decreased CTSS activity(P<0.05)while had no significant effect on CTSS protein abundance(P>0.05). caspase 3 protein abundance kept stable(P>0.05)and caspase 3 activity fluctuated in the process of IVM(P<0.05). 5μmol/L E-64 and 10μmol/L E-64 decreased caspase 3 activity in different levels(P<0.05;P<0.01)and 10μmol/L E-64 up-regulated caspase 3 protein abundance(P<0.01),whileμmol/L E-64 had no significant effects on caspase 3 protein abundance(P>0.05). In lamb oocytes, CTSB activity increased(P<0.01)while protein abundance kept stable(P>0.05). 5μmol/L E-64 had no significant effects on CTSB activity while decreased protein abundance very significantly(P<0.01). 10μmol/L E-64 decreased CTSB activity(P<0.05)and increased CTSB protein abundance(P<0.01). CTSS activity and protein abundance kept stable(P>0.05), 5μmol/L E-64 and 10μmol/L E-64 significantly decreased CTSS protein abundance(P<0.05;P<0.01),and 5μmol/L E-64 decreased CTSS activity(P<0.05), while it had no significant changes in 10μmol/L E-64 group(P>0.05). caspase 3 activity and protein abundance kept stable(P>0.05), 5μmol/L E-64 significantly increased caspase 3 activity and protein abundance(P<0.05), while caspase 3 activity and protein abundance had no significant changes in 10μmol/L E-64 group(P>0.05).