The Study On Molecular Cloning Of Sheep CTSB And Relativity Between Its Activity And Developmental Capacity Of Oocytes

Cathepsin B (CTSB), an important lysosomal cysteine protease, plays a role invarious physiological and pathological processes, and has an important effect on the abilityof oocytes maturation and embryos development. Studying on the change regularity ofCTSB activity of sheep oocytes during in vitro maturation (IVM), and the relativity withdevelopmental capacity of oocytes, could provide a theoretical basis and technicalmeasures to improve the efficiency of sheep embryo production in vitro.The amplified CDS sequence of CTSB from sheep, a total length of1008bp, coding335amino acids, had been submitted to Genbank (accession number: KC699918). Using avariety of bioinformatics software predicted that sheep CTSB protein had the typicalstructural characteristics of papain-like cysteine proteases, consisting of signal peptide,propeptide and mature peptide. It had three cysteine protease active sites: Cys108, His278andAsn298, and also a CTSB protein specific characteristic “occluding loop”. It also hadmodification of phosphorylation and glycosylation after translation.This experiment measured the CTSB activity change regulation in sheep cumulusoocyte complexes (COCs) during IVM using microplate reader by the fluorescent substratemethod. The results indicated that the CTSB activity of sheep COCs showed a decliningtrend with the extension of IVM time. Comparing with the RFU ratio (1.000) of CTSBactivity of adult sheep at IVM0h, it dropped to0.7740at IVM8h (P<0.05),0.7030atIVM16h (P<0.01) and0.3709at IVM24h (P<0.01). In juvenile sheep group, comparedwith the RFU ratio at IVM0h (1.0228), there was no significantly change at IVM8h(1.0267)(P>0.05), but it dropped to0.7817at IVM16h (P<0.01),0.6087at IVM24h(P<0.01). At IVM8h and24h, the CTSB activity of juvenile sheep COCs wassignificantly higher than adult sheep. Density mean of CTSB activity in sheep oocytes andcumulus cells were tested using laser scanning confocal microscope. The results showedthat compared with the density mean of CTSB activity in oocytes at IVM0h (0.0119), itwas extremely significantly decreased at IVM24h (0.0046)(P<0.01). And CTSB activityin cumulus cells at IVM24h (0.2175) was significantly decreased than IVM0h (0.2960)(P<0.05). The density mean of CTSB activity in oocytes and cumulus cells of poor qualitygroup (0.0439and0.4371) were extremely significantly higher than the normal quality group after IVM24h (P<0.01).E-64is one kind of cysteine protease inhibitors. Studying on the relativity betweenCTSB activity inhibited by different concentrations E-64and developmental competenceof oocytes showed that CTSB activity of adult sheep COCs inhibited by1μmol/L E-64atIVM8h was significant lower than the control group (P<0.05), and the5μmol/L and10μmol/L E-64groups were extremely significant lower than control (P<0.01). The CTSBactivity at IVM16h, all the E-64groups were extremely significant lower than control(P<0.01), and at IVM24h significant lower than control (P<0.05). The CTSB activity ofjuvenile sheep COCs inhibited by5μmol/L E-64at each time points during IVM wasextremely significantly decreased than control (P<0.01). There was nosignificantly reduced of CTSB activity in adult sheep oocytes of1,5,10μmol/L E-64groups at IVM24h (P>0.05), but CTSB activity in cumulus cells appeared significantdecreased (P<0.05).1,5,10μmol/L E-64had no significant effect on adult sheep oocytesmaturation rates and cleavage rates (P>0.05), but39.8%~48.6%increased in the blastocystrates (P<0.05).