At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.

Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, we observe that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min postinsemination. Further, Ca(2+) was both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca(2+) ionophore A23187 caused MAP kinase inactivation and triggered DNA synthesis. When the rise in intracellular Ca(2+) was inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remained high. Finally, inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggered DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis.

Lymphogranuloma venereum (LGV)-caused by Chlamydia trachom atis serovars Ll , L2, or L3-rarely occurs in the United States. The disease clinically manifests in three stages: primary, secondary, and tertiary. Theprimary manifestation, a self-limited genital ulcer at the site of inoculation, often is absent by the time the patient seeks medicalattention. Themostcommon clinical manifestation of LGV isevident in its secondary stage: unilateral tender inguinal and/or femoral lymphadenopathy. However, proctocolitis or inflammatory involvement of perirectal or perianal lymphatic tissues resulting in fistulas and strictures may also occur. The diagnosis of LGV is usually made serologically and by exclusion of other causes of inguinal lymphadenopathy or genital ulcers. Doxycycline is thepreferred treatment; it cures the infection and prevents ongoing tissue damage. This case highlights an unusualmanifestation ofLGVinfection-cervica llymphadenopathy following suspected oropharyngeal infection with C trachomatis. Head and neck manifestations of LGV may become an increasing problem in the future if sexual practices such as orogenital contact become more widespread.