ResearchFusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca 2+ , leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca 2+ oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCζ) has been identified in mice. Mouse PLCζ triggers Ca 2+ oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCζ homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCζ protein sequences indicates a shorter X-Y linker region in human PLCζ and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCζ elicits Ca 2+ oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCζ elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCζ from monkeys and mice. These results are consistent with the proposal that sperm PLCζ is the molecular trigger for egg activation during fertilization and that the role and activity of PLCζ is highly conserved across mammalian species.

Reproduction 124, 611-623). PLC may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLC is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLC structure-function, we assessed the ability of PLC and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca 2؉ changes in intact mouse eggs. PLC and the closely related PLC␦1 had similar K m values for phosphatidylinositol 4,5-bisphosphate, but PLC was around 100 times more sensitive to Ca 2؉ than was PLC␦1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLC constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca 2؉ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca 2؉ oscillations. These data suggest that the exquisite Ca 2؉ -dependent features of PLC regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca 2؉ oscillations in intact mouse eggs.

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lllinois 60566 (F.W., J.P., R.C., C.S.)3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMCR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The ratelimiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMCR activity could be limiting in plants, we introduced a constitutively expressing hamster HMCR gene into tobacco (Nicotiana tabacum L.) plants to obtain unregulated HMCR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMCR gene led t o a 3-t o 6-fold increase in the total HMCR enzyme activity. Total sterol accumulation was consequently increased 3-to 1 O-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. l h e leve1 of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMCR activity, these results indicate that the activity of one or more later enzyme(s) i n the pathway must also be involved i n determining the relative accumulation of end-product sterols.The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.