273 EXPRESSION PATTERN OF CONNEXIN 43 (Cx43) AND POLY-(A) POLYMERASE (PAP) GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED IN VITRO

Abstract

Polyadenylation of pre-mRNA is carried out by poly-(A) polymerase (PAP), and study of the transcription pattern of this gene is said to indicate the developmental competence of the embryos. Connexin 43 (Cx43) is one of the important gap junction proteins that controls growth, cellular differentiation, and embryonic development. The objective of the present investigation was to study the expression pattern of PAP and Cx43 genes in buffalo (Bubalus bubalis) embryos produced in vitro. Embryos were produced from the slaughterhouse ovaries using standard IVMFC protocol (Rajhans et al. 2006 J. Reprod. Fertil. Dev. 18, 253–254). Briefly, oocytes were aspirated from follicles (2–8 mm in diameter) and matured in vitro in TCM-199 supplemented with 10% FCS and epidermal growth factor (20 ng mL^-1) for 24 h. Presumptive zygotes after 18 h of fertilization were cultured in mSOF containing insulin-like growth factor-1 (100 ng mL^-1) and β-mercaptoethanol (100 µM) for 9 days or until blastocyst formation, whichever was earlier. Pools of immature (n = 200), in vitro-matured (n = 200), oocytes and embryos (2–4 cell, n = 83; 8–16 cell, n = 80; morula, n = 77), and blastocysts (n = 40) were collected for mRNA isolation. Immature and in vitro-matured oocytes were treated with 1X trypsin-EDTA solution to remove the attached cumulus and then washed with TCM-199 before mRNA isolation to avoid any contamination of these cells during RNA isolation. mRNA from each pool was isolated using a commercially available direct mRNA isolation kit (Oligotex Direct mRNA kit; Qiagen, Valencia, CA, USA). cDNA was prepared using specific reverse primer and M-MLV RT in 20 µL reaction volume following manufacturer's instructions. Polymerase chain reaction was done for 35 cycles with annealing temperatures of 60°C and 58°C for 252 bp of PAP and 425 bp of Cx43, respectively. Amplicons were subjected to restriction endonuclease digestion for further confirmation of expressed genes. RT-PCR amplicon of PAP was digested with HaeIII to obtain characteristic band patterns at 119 bp and 133 bp, and Cx43 RT-PCR amplicon was digested with EcoR1 to obtain characteristic band patterns at 137 bp and 288 bp. While PAP expression could be detected in all stages of developing embryos starting from immature oocytes to blastocyst stage, Cx43 mRNA was detected in immature oocytes to morula stage but not in blastocyst-stage embryos. It could be concluded that the expression patterns of PAP and Cx43 genes in buffalo embryos produced in vitro are similar to those of cattle embryos.