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.

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 ...

One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization.

Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCζ), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCζ was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.

Background: During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes.