Developmental enhancement of intracytoplasmic sperm injection (ICSI) - Generated quail embryos by phospholipase C zeta cRNA

摘要

The present study was conducted to improve the rate and stage of blastoderm and embryo development of quail oocytes by intracytoplasmic sperm injection (ICSI) with quail phospholipase C zeta (PLC zeta) cRNA. Blastoderm development of quail oocytes cultured for 24 or 72 h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLC( cDNA clone was isolated from sperm by RT-PCR. The 1978 bp nucleotide sequence contained an ORF encoding 637 amino acids of protein. The deduced quail PLC zeta protein consists of EF-hand domain, X and Y catalytic domain and C2 domain, but lacked a plextrin-homology (PH) domain at the N-terminus. RT-PCR analysis revealed that PLC zeta mRNA is expressed only in the testis. ICSI into a quail oocyte without PLC zeta cRNA induced blastodermal development in 16% (4/25) of the oocytes which reached stages III-VI 24 h after culture. In contrast, ICSI together with PLC zeta cRNA (in 3 nl at 60 mu g/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.06: 7/19) with embryos reaching stages VI-VII Furthermore, 72 h after culture ICSI into a quail oocyte without PLC zeta cRNA induced embryo development in 15.4% (2/13) of the oocytes and reached stages V-VII. In contrast, ICSI together with PLCC cRNA (in 3 nl at 60 mu g/ml) into a quail oocyte induced 3 fold more embryo development (50516, 9/18) with embryos between stages VI and over X. The microinjection of PLC zeta cRNA significantly improved the conventional ICSI method by enhancing the proportion and the stages of embryo development. Accordingly, the present PLC zeta cRNA microinjection method in birds may contribute to assist in the production of transgenic birds and protection of endangered species of birds.