135 EXPRESSION OF APOPTOSIS-RELATED GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED THROUGH IN VITRO FERTILIZATION AND PARTHENOGENETIC ACTIVATION

Abstract

Apoptosis, a highly conserved evolutionary mechanism that allows an organism to tightly control cell numbers, tissue size, and protect itself from dangerous cells and unfavourable environments that threaten homeostasis, is generally directed by specific genes involved in the regulation of a series of pro-apoptotic (BAX) and anti-apoptotic (BCL-XL) proteins that are expressed during early development. All mammalian species show the highest level of spontaneous apoptotic processes at the blastocyst stage. These proteins prevent apoptosis by maintaining the cell survival by interfering with the release of cytochrome-C from mitochondria. In this study, immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 μg mL^–1 porcine FSH for 24 h in a CO₂ incubator (5% CO₂, 90 to 95% relative humidity) at 38.5°C. The mature oocytes were used for IVF, and the cleaved embryos were cultured for 8 days in culture medium (CR2 medium containing 0.6% BSA and 10% FBS) for production of embryos at different stages. The parthenotes were produced with exposure of 7% ethanol, 6-dimethyl aminopurine and cultured for 8 days in culture medium. The total RNA was isolated from oocytes and embryos and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA), according to manufacturer protocol. The PCR cycle included heating to 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60 (BAX) and 62°C (BCL) for 30 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The amplified product of both genes were separated on agarose gel and densitometry data for band intensities were generated using AlphaDigiDocTM AD-1201 software under a Windows^TM environment and data analysed with the help of SYSTAT software. Relative abundance of BCL-XL transcripts in immature, mature oocytes and embryos produced through IVF (i.e. 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stage) were 25.33 ± 0.90, 12.67 ± 1.20, 37.67 ± 0.90, 30.67 ± 0.30, 23.67 ± 0.90, 18.33 ± 0.90, and 27.00 ± 1.20, respectively, whereas in parthenogenesis these values were 23.67 ± 0.88, 13.67 ± 1.20, 23.67 ± 1.20, 22.34 ± 0.88, 24.34 ± 0.88, 33.67 ± 0.88, and 45.34 ± 1.20, respectively. Relative abundance of BAX transcripts by IVF were 23.0 ± 0.60, 0.33 ± 0.10, 4.00 ± 0.60, 5.00 ± 0.60, 0.37 ± 0.06, 13.0 ± 0.66, and 56.7 ± 0.90; and by parthenonenesis were 22.3 ± 0.90, 0.13 ± 0.03, 13.67 ± 0.90, 14.0 ± 0.60, 15.33 ± 0.90, 64.67 ± 2.20, and 55.0 ± 2.10, respectively. In conclusion, the expression pattern of the apoptosis-related genes revealed that the incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages such as immature oocytes, morula, and blastocyst than the early cleavage stage embryos.