Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs.
Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.
Kouchi
Zen
Z
Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. zkouchi@research.twmu.ac.jp
Fukami
Kiyoko
K
Shikano
Tomohide
T
Oda
Shoji
S
Nakamura
Yoshikazu
Y
Takenawa
Tadaomi
T
Miyazaki
Shunichi
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007313:Insecta; D007477:Ions; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D009991:Oscillometry; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013094:Spermatozoa / Q000378:metabolism; D013997:Time Factors; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*
D018076:DNA, Complementary; D007477:Ions; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D012313:RNA; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse; D002118:Calcium