311 EXPRESSION OF PLURIPOTENT MARKER NUCLEOSTEMIN IN BUFFALO (BUBALUS BUBALIS) EMBRYOS AND EMBRYONIC STEM CELLS GENERATED THROUGH PARTHENOGENETIC ACTIVATION

Abstract

Nucleostemin is a newly found putative GTPase protein that binds to P53 and exists mainly in the nucleoli and at a very low level in nucleoplasm of undifferentiated embryonic stem cells (ESC) and myeloid stem cells but is not expressed in committed and terminally differentiated cells. Embryonic stem cells are pluripotent cells derived from the inner cell mass (ICM) of blastocysts. The ICM and ESC express a number of transcription factors, and their expression is used as a pluripotency marker in the ESC of many species. The present study was undertaken to identify expression of the nucleostemin gene in different developmental stages of buffalo embryos and cultured ESC. Parthenogenetic activation is a process by which an oocyte can be developed up to blastocyst without fertilization. The parthenotes were produced by following protocol. Briefly, immature oocytes were aspirated from slaughterhouse buffalo ovaries and subjected to in vitro maturation for 24 h in a CO₂ incubator (5% O₂, 5% CO₂, 90–95% relative humidity) at 38.5°C. After 24 h of in vitro maturation, oocytes were 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, and they were then subjected to in vitro culture. The activated embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS to obtain different stages (immature and mature oocytes 2-, 4-,8–16-cell, morula, and blastocyst) of embryos. A total of 23 blastocysts were produced parthenogenetically, of which 5 blastocysts were used for nucleostemin expression and the rest were used for ICM isolation. The isolated ICM were subsequently cultured on mitomycin-C (10 μg mL^–1) treated buffalo fetal fibroblast feeder layer in DMEM medium supplemented with 20% fetal bovine serum, 1 000 IU mL^–1 of mouse leukemia inhibitory factor, 1% nonessential amino acids, 2 mM L-glutamine, and 50 μg mL^–1 gentamycin. These ESC were cultured up to 5 passages. The 5 embryos of different developmental stages and a clump of ESC were used for nucleostemin expression. The total RNA was isolated and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA) according to manufacturer protocol. To amplify the nucleostemin gene, the PCR cycle was carried out and included heating to 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s. The expressions of nucleostemin transcript were observed in all the developmental stages including immature and mature oocytes. The transcript was highly expressed in the 2-cell stage, blastocysts, and ESC, but immature oocytes and 8–16-cell stage showed lower expression. The experiment was repeated, and the same result was found. To our knowledge this is the first report in buffalo. It is concluded that the transcript was expressed in all the early stages of parthenogenetically derived buffalo embryos from immature oocytes to blastocysts and continued to be expressed in ESC.