Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca 2+ concentration ([Ca 2+ ] i ). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca 2+ ] i oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca 2+ ] i oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca 2+ ] i oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.

Reviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca(2+)) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel in Ca(2+) waves and Ca(2+) oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62-78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254] and the different temporal responses of Ca(2+) in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176]. Those reviews raise the importance of identifying how Ca(2+) causes the events of egg activation (EEA) and to what extent these temporal Ca(2+) responses encode developmental information. This review covers recent studies that have analyzed how these Ca(2+) signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca(2+), and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca(2+) and the EEA, as well as discuss how oscillatory or multiple Ca(2+) signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca(2+), Ca(2+)-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species.

2+] i ) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP 3 R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca 2+ release. IP 3 R1-mediated Ca 2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca 2+ ] i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP 3 R1 function in eggs. Using mouse and Xenopus eggs, we show that IP 3 R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP 3 R1 at at least one highly conserved site, and that its mutation abrogates IP 3 R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP 3 R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca 2+ ] i oscillations in response to agonists and show compromised IP 3 R1 function. These findings identify IP 3 R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP 3 R1 function in eggs that serves to optimize [Ca 2+ ] i release at fertilization. KEY WORDS: Fertilization, Ca 2+, IP3R1, Mouse, MAPK, Xenopus Development 133, 4355-4365 (2006) Jones and Whittingham, 1996). Given that during maturation and after activation/fertilization the changes in IP 3 R1 concentrations and content of the Ca 2+ stores are small (Brind et al., 2000;Iwasaki et al., 2002;Jellerette et al., 2000), it is likely that other mechanisms might regulate IP 3 R1 function in eggs.Phosphorylation has been shown to be an important regulatory mechanism of IP 3 R1 function (Bezprozvanny, 2005;Patterson et al., 2004a). Among the protein kinases that phosphorylate IP 3 R1 are: protein kinase A and protein kinase C (Ferris et al., 1991;Vermassen et al., 2004a); protein kinase G (Koga et al., 1994); Ca 2+ /calmodulindependent protein kinase II (Ferris et al., 1991;Zhu et al., 1996); the tyrosine kinases Fyn (Jayaraman et al., 1996) and Lyn (Yokoyama et al., 2002); Rho kinase (Singleton and Bourguignon, 2002); and, very recently, protein kinase B (Khan et al., 2006) (V.V., H.D.S. and J.B.P., unpublished). In most cases, IP 3 R1 phosphorylation by these kinases enhances Ca 2+ conductivity, but none of these kinases appears to be intimately associated with cell-cycle transitions. Most importantly, abrogation of their activities by pharmacological inhibitors does not affect IP 3 R1 function in eggs (Carroll and Swann, 1992;Smyth et al., 2002;Swann et al., 1989). However, a ...