Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes

Background & objectives: The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes. Methods: All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ?6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential. Results: Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-?, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group. Interpretation & conclusions: The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 ?M roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Activated charcoal fi lter effectively reduces p-benzosemiquinone from the mainstream cigarette smoke and prevents emphysema.

In this paper, we have made a comparative evaluation of the cytotoxicity and pathophysiological effects of mainstream smoke from cellulose acetate (CA)-fi ltered cigarettes with that of charcoal-fi ltered cigarettes developed in our laboratory. Previously, we had demonstrated that the mainstream smoke from an Indian CA-fi ltered commercial cigarette contains p-benzosemiquinone (p-BSQ), a major, highly toxic, long-lived water-soluble radical. Here, we have examined 16 brands of different CA-fi ltered cigarettes including Kentucky research cigarettes, and observed that mainstream smoke from all the cigarettes contains substantial amounts of p-BSQ (100–200 μg/cigarette). We also show that when the CA fi lter is replaced by a charcoal fi lter, the amount of p-BSQ in the mainstream smoke is reduced by 73–80%, which is accompanied by a reduction of carbonyl formation in bovine serum albumin to the extent of 70– 90%. The charcoal fi lter also prevented cytotoxicity in A549 cells as evidenced by MTT assay, apoptosis as evidenced by FACS analysis, TUNEL assay, overexpression of Bax, activation of p53 and caspase 3, as well as emphysematous lung damage in a guinea pig model as seen by histology and morphometric analysis. The results indicate that the charcoal fi lter developed in our laboratory may protect smokers from cigarette smoke-induced cytotoxity, protein modifi cation, apoptosis and emphysema.