SIVA KUMAR, A.V.N (Major)Rao, V.HPadmaja, KDEEPA, P2016-12-152016-12-152015-11http://krishikosh.egranth.ac.in/handle/1/90361ABSTRACT: To ascertain whether the support provided by Leptin to the PFs’ in culture (Kamalamma et al., 2015) was due to its ability to modulate apoptosis related genes, the present study is undertaken. Accordingly in the present study expression levels of two test genes BCl2 (antiapoptotic), BAX (apoptotic) and three reference genes viz., 18S rRNA, HPRT1, RPLPO were studied in the Cumulus cells and Oocytes during in vivo and in vitro folliculogenesis in sheep. Sheep ovaries were collected from the local slaughter house. The Cumulus cells and Oocytes from different development stages of ovarian follicles i.e., preantral, early antral, antral and large antral follicles were isolated. Preantral follicles (PFs’) isolated from the ovaries were cultured in a medium containing 10 ng/ml of recombinant human Leptin for 3 minutes, 2, 4, or 6 days to obtain cultured preantral, early antral, antral and large antral follicles respectively. Furthur some COCs in in vivo and in vitro grown large antral follicles were matured in vitro for 24h. The expression pattern of the test and reference genes (18SrRNA, HPRT1, RPLPO) in the Cumulus cells and Oocytes was studied separately at the different development stages mentioned earlier by Quantitative Reverse transcription Real time PCR (qRT-PCR). Quantitative expression of BCl2 gene in the Cumulus cells isolated from different in vivo grown ovarian follicles was significantly lower in the pre antral and early antral follicles than corresponding stages of follicles cultured in Leptin. However, the expression was significantly higher in the in vivo grown antral, large antral and COCs from large antral follicles matured in vitro for 24h. Quantitative expression of BCl2 in the Oocytes isolated from the pre antral follicles and COCs in large antral follicles matured in vitro for 24h was significantly lower than their corresponding Leptin cultured stages. However, there was no significant difference in the BCl2 expression in the Oocytes in early antral, antral and large antral follicles compared to their corresponding Leptin cultured stages. Quantitative expression of BAX in the Cumulus cells isolated from in vivo grown ovarian follicles was significantly lower in the preantral follicles and Cumulus cells from COCs after in vitro maturation for 24h than their corresponding Leptin cultured stages. However, the Cumulus cells from in vivo antral follicles exhibited a significantly higher level of expression than corresponding Leptin cultured follicles. Quantitative expression of BAX in the Oocytes isolated from the in vivo grown antral follicle and Oocytes from COCs after in vitro maturation for 24h was significantly lower than the corresponding in vitro stage. However, Oocytes from large antral follicles expressed BAX to a significantly higher level than corresponding in vitro stage. The expression pattern of BAX in the Oocytes isolated from the different in vivo grown ovarian follicles was similar to the corresponding in vitro grown stages in early antral and large antral follicles. From the present study it is concluded that supplementation of Leptin (10 ng/ml) in the culture medium induced a different quantitative expression pattern of BCl2 in Cumulus cells and Oocytes than in vivo grown ovarian follicles. Leptin inclusion in the culture medium was able to simulate the in vivo pattern of BAX gene expression in the Cumulus cells and Oocytes at some but not all of the development stages.enSheep; Leptin; BCl2; antiapoptotic; BAX ; apoptotic; genes; folliculogenesis; 18S Rrna; HPRT1; RPLPOThesis