165 EXPRESSION OF Oct-4 IN BUFFALO (BUBALUS BUBALIS) EMBRYOS GENERATED THROUGH IN VITRO FERTILIZATION OR PARTHENOGENETIC ACTIVATION

Abstract

Octamer-4 (Oct-4) is a member of Class V of the POU transcription factors family, which is involved in transcriptional regulation during early embryonic development and cell differentiation. It is expressed in the inner cell mass of blastocysts and in embryonic stem cells (ESCs), and its expression is widely used as a marker of pluripotency in ESCs in many species. This study was, therefore, carried out to examine the expression of Oct-4 in embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages generated through IVF or parthenogenetic activation. A total of 100 embryos were used in the study, 10 for each embryonic stage from both methods of embryo production. Immature oocytes obtained from slaughterhouse buffalo (Bubalus bubalis) ovaries were subjected to IVM in TCM-199 + 10% FBS + 5 µg mL^–1 pFSH + 0.81 mm sodium pyruvate for 24 h in a CO₂ incubator (5% O₂, 5% CO₂, 90–95% relative humidity) at 38.5°C. IVF was carried out immediately after IVM; the cleaved embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS for production of embryos at different stages. For production of embryos through parthenogenesis, after 24 h of IVM, oocytes were denuded of cumulus cells by incubation in 0.2% hyaluronidase in Dulbecco's phosphate-buffered saline for 2 min. The denuded oocytes with a prominent polar body were parthenogenetically activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mm 6-dimethyl aminopurine in CR2 medium for 3.5 h in a CO₂ incubator (5% O₂, 5% CO₂, 90–95% relative humidity) at 38.5°C, and then subjected to IVC as described above. A two-step RT-PCR was carried out using Cells-to-cDNA Kit-II (Ambion, Austin, TX, USA), using bovine primers 52-GTT CTC TTT GGA AAG GTG TTC-3' and 5'-ACA CTC GGA CCA CGT CTT TC-3' for the amplification of Oct-4. For this, the embryos were washed with PBS, transferred to 30 µL of cold cell lysis buffer and incubated in a thermal cycler at 75°C for 10 min. The cell lysate was treated with DNase-I at 37°C for 30 min to degrade genomic DNA and then heated at 75°C for 5 min to inactivate the DNase-I. The cell lysate (10 µL) was used for making cDNA using random primer. The PCR cycle included heating to 94°C for 2 min, followed by 33 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s. A final extension at 72°C for 10 min was carried out to complete the amplification of the Oct-4 gene. Transcripts of Oct-4 were detected at all of the embryonic stages, from the 2-cell through the hatched blastocyst stage in both IVF and parthenogenetically generated embryos. These results indicate that Oct-4, which is believed to be a reliable marker for pluripotency of ESCs in a number of species, is expressed in early cleavage-stage buffalo embryos and continues to be expressed in preimplantation-stage blastocysts.