1.Primary Study on the Effect of Resveratrol on Early Ovarian Follicle Development and Oocyte Apoptosis in Rat 2.cDNA Cloning and Expression of Trichomonas Vaginalis RRas Gene
|Keywords||Resveratrol Follicular development Apoptosis TUNEL Rats Trichomonas vaginalis RRAS gene Clone Prokaryotic expression|
Objective To study the impact of resveratrol on rat early follicular development and oocyte apoptosis. Materials and Methods adult Sprague-Dawley (SD) rats were caged, newborn rats were randomly divided into three groups: control group, female rats fed group, intraperitoneal injection group. Control group of normal birth 1d, 2d and 4d age rats; female rats fed group SD female rats in a normal pregnancy pregnant 12d began resveratrol (25mg/kg · d) orally, until delivery, the newborn rats removal of ovaries in 1D, 2d and 4d age; intraperitoneal injection group for normal rats born within 12h after birth, intraperitoneal injection of resveratrol (25mg/kg · d), whichever is the ovarian in 2d, 4d age. Taken ovaries were fixed, dehydrated, embedded slices, hematoxylin - eosin (hematoxylin-eosin, HE) staining and terminal deoxynucleotidyl acyl transferase-mediated dUTP nick end labeling method (TdT-mediated dUTP Nick -End Labeling, TUNEL) observed resveratrol on rat early follicular development and apoptosis. 1d age rat dams fed group oocyte nest oocytes ratio of 69.42%, 82.49% lower than in the control group; its primordial follicle ratio of 30.47%, 18.13% higher than that in the control group, the differences were There are statistically significant (p <0.05). 2d age rat dams fed group and intraperitoneal injection of oocytes nest proportion of oocytes were 53.20% and 39.78%, significantly higher than the 15.76%; primordial follicles developing follicles proportion were 46.40% and 4.51%, 57.64% and 2.58%, 74.57% and 9.67% lower than the control group, the differences were statistically significant (p <0.05). 4d Age rat maternal intragastric administration and intraperitoneal injection group oocyte nest oocytes proportion of 5.39% and 9.39%, significantly higher than 2.62%, the difference was statistically significant (p <0.05) ; primordial follicles, the proportion of developing follicles were 63.22% and 30.77%, 66.50% and 24.11%, respectively, compared with the control group, the difference was not statistically significant (p> 0.05). 1d age female rats fed rats oocyte apoptosis rate was 10.29%, compared with 4.43% of the control group, the difference was statistically significant (p <0.05). 2d age of female rats fed group and intraperitoneal injection of follicular apoptosis rates were 9.88% and 12.95%, respectively, the control rats follicle apoptosis rate of 8.50%, the difference was not statistically significant (p> 0.05). Conclusion resveratrol can delay the rat oocyte nest breakdown, inhibit the development of primordial follicles start; resveratrol may promote apoptosis of oocytes. The research purposes cloning and expression of the gene the Trichomonas vaginalis RRAS (TvRRas,), in order to further investigate the localization and function. Materials and methods from Trichomonas vaginalis cDNA expression library constructed the amplification TvRRas gene fragments by PCR and cloned into pGEM-T vector by PCR, restriction enzyme digestion and sequencing was subcloned to the prokaryotic expression vector pET-41a, transforming Escherichia coli BL21 (Ecoli), inducible by isopropyl-beta-D-galactoside (IPTG), nickel - nitro triacetic acid (Ni-NTA) purified expression product by SDS-polypropylene amide gel electrophoresis (SDS-PAGE) analyzes. Results from amplification of Trichomonas vaginalis cDNA expression library the TvRRas gene fragment length 522bp, restriction enzyme digestion and DNA sequencing confirmed that pET-41a/TvRRas recombinant plasmid constructed correctly, expression of the fusion protein molecular weight of about An expected 45kDa. Conclusion The construction prokaryotic expression plasmid pET-41a/TvRRas, and can be expressed in E. coli, the fusion protein molecular size expected.