Temporal expression pattern of insulin-like growth factors (IGF-1 and IGF-2) ligands and their receptors (IGF-1R and IGF-2R) in buffalo (Bubalus bubalis) embryos produced in vitro

摘要

The objective of the study was to assess the temporal expression of genes for IGF-1, IGF-2 and their receptors in different pre-implantation stage buffalo embryos (from oocytes to blastocyst) and to evaluate the effect of IGF-1 on culture media during in vitro buffalo embryo development. Buffalo oocytes were retrieved from slaughterhouse derived ovaries. In vitro matured oocytes were fertilized with frozen buffalo bull semen. Presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF supplemented with beta-mercaptoethanol (100 mu M) and without (control) or with IGF-1 (100 ng/ml) till blastocyst stage. For each treatment five replicates were taken. Supplementation of IGF-1 to embryo culture media resulted in significantly higher blastocyst formation rate (29.7% versus 25.6%; p < 0.05) than the control group. In vitro produced embryos of different stages were stored in small amount of PBS at -70 degrees C till used for RNA isolation. Total RNA isolated from oocytes and pre-implantation embryos was reverse transcribed to cDNA in total volume of 25 mu l reaction mixture using Moloney-Murine Leukemia Virus Reverse Transcriptase (MMLV-RT). Polymerase chain reaction (PCR) was performed using cDNA from different pools of oocytes and embryos. RT-PCR revealed that IGF-1 mRNA was neither expressed in oocytes (immature, matured and matured vitrified oocytes) nor at any pre-implantation stage embryos while mRNA for IGF-1R, IGF-2 and IGF-2R were detected in all oocytes as well as in all preimplantation stage embryos. It can be concluded that supplementation of IGF-1 is useful for in vitro oocyte maturation and embryo development in buffalo. (c) 2010 Elsevier B.V. All rights reserved.