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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_i oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca²⁺]_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 [Ca²⁺]_i oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

We have previously demonstrated that initiation of intracellular calcium ([Ca2+]i) oscillations in mouse eggs signals activation or apoptotic death d

During meiotic maturation, numerous cytoplasmic and nuclear events take place that prepare the oocytes for fertilization. These changes are initiated by an increase in the activity of several kinases, most notably maturation-promoting factor, also called histone H1 kinase. Another kinase, mitogen-activated protein (MAP) kinase, is also stimulated during this period. In this study, we investigated the role of MAP kinase in bovine oocyte maturation. First, the kinetics of activation of histone H1 and MAP kinases during maturation were assessed simultaneously by evaluating their catalytic activities in vitro. We found that they are activated at approximately the same time, around germinal vesicle breakdown (GVBD). Then, at approximately 15 h of maturation, the activity of H1 kinase temporarily decreases, whereas MAP kinase remains high through the metaphase II stage. Second, the activation and catalytic activity of MAP kinase was directly evaluated by Western blotting and by an in-gel kinase assay. We determined that MAP kinase becomes activated and exhibits a decreased mobility through SDS-polyacrylamide gels, and that its catalytic activity increases as maturation progresses. In our system, most of the MAP kinase activity can be attributed to p42MAPK2. Third, the activation pathway of MAP kinase was explored. In Xenopus oocytes, MAP kinase is activated by a kinase cascade that includes several upstream activators; one of them is the product of the proto-oncogene mos. In bovine oocytes, injection of Mos RNA elicited a rapid maximal activation of MAP kinase that resulted in accelerated resumption of meiosis and GVBD. These results were thought to be mediated by an expression of a kinase-active Mos RNA failed to activate MAP kinase. Together, these results suggest a role for MAP kinase during the initiation and progression of meiosis in bovine oocytes. The data also suggest the presence of an MAP kinase-activating cascade that can be initiated by the Mos protein.

ICSI bypasses not only fusion of the gametes but also a series of signalling events that occur in the sperm prior to and during interaction with the oocyte’s vestments. The effect of this altered encounter of the gametes on the fertilization‐associated intracellular calcium ([Ca²⁺]_i) oscillations has not been thoroughly investigated. Here, ICSI and IVF were performed using gametes from two mouse strains, B6D2F1 and CD1, and in‐vitro development, pattern of [Ca²⁺]_i oscillations and down‐regulation of inositol 1,4,5‐trisphosphate receptor‐1 (IP3R‐1) in the produced embryos were compared. ICSI and IVF resulted in comparable rates of activation and pre‐implantation development. However, ICSI‐generated zygotes cleaved at a slower rate, had lower cell numbers and lower hatching rates. The deleterious effects of ICSI could not be exclusively attributed to the injury by the injection since sham‐injected IVF zygotes only exhibited delayed progression to the blastocyst stage. ICSI and IVF induced similar initial [Ca²⁺]_i responses, although ICSI zygotes exhibited shorter durations of [Ca²⁺]_i oscillations and showed diminished degradation of IP3R‐1. Importantly, sperm manipulation affected the pattern of oscillations, which further decreased pre‐implantation developmental rates. Our results demonstrate that ICSI‐induced [Ca²⁺]_i responses are not equivalent to those initiated by IVF and that this may have developmental consequences.