Developmental Enhancement of Intracytoplasmic Sperm Injection (ICSI)-Generated Quail Embryos by Phospholipase C(zeta) cRNA

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 72h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLC(zeta) cDNA clone was isolated from sperm by RT-PCR. The 1978bp 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 24h after culture. In contrast, ICSI together with PLC(zeta) cRNA (in 3nl at 60μg/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.8%, 7/19) with embryos reaching stages VI-VII. Furthermore, 72h 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 PLC(zeta) cRNA (in 3nl at 60μg/ml) into a quail oocyte induced 3 fold more embryo development (50%, 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.