IP3R Agonists Sensitize CICR
A sensitized CICR mechanism is critical for the persistence of [Ca2+]i oscillations in fertilized mammalian eggs, although the molecular mechanism(s) responsible for sensitizing the CICR is not known. Because SF stimulates production of IP3, increased [IP3]i may be responsible for sensitizing CICR. Our finding that injection of adenophostin A, which shares structural motifs with IP3, considerably enhanced Ca2+ release in response to injection of CaCl2 suggests that elevated [IP3]i alone might be capable of sensitizing CICR. Because adenophostin A is a nonhy-drolysable IP3R agonist, it is presumed that its injection resulted in steady intracellular levels of the agonist. Whether [IP3]i oscillates with each [Ca2+]i rise during fertilization is not known.
Another possibility is that the sperm/SF may carry an activator of the egg PLCs. Mammalian eggs express PLC|31, PLC(33, PLC71, and PLC72, and because of their much greater volume, they are likely to contain in excess of 1000-fold greater amounts of the isoforms present in sperm. In addition, the egg PLCs are easily activated to produce [Ca2+]i oscillations. For example, [Ca2+]i rises are initiated in mammalian eggs after injection of GTP7S or when eggs expressing foreign cell-surface receptors coupled to G-proteins or tyrosine kinase pathways are stimulated with appropriate agonists. Thus, it appears unlikely that SF would induce [Ca2+]i oscillations without involving the egg PLCs.
How SF May Activate the PI Pathway
The mechanism by which the sperm/SF stimulates the PI pathway remains unresolved. Of the 10 mammalian PLC isozymes identified to date, mammalian sperm express PLCpl, PLC7I, and PLC72. In addition, mouse testis expresses high levels of PLC81 mRNA, and this mRNA appears confined to spermatogonia cells. Further, two splicing variants of PLC84, ALTI and ALTII, are abundantly expressed in rat testis. Based on these reports and on additional functional studies, it has been proposed that a PLC is the active component of SF. Subsequent studies, however, showed that addition/injection of several recombinant PLCs and tissue extracts containing native PLCs into sea urchin egg extracts and mouse eggs failed to trigger Ca2+ release despite the fact that the specific PLC activity of recombinant PLCs in in vitro assays was significantly higher than SF PLC activity.
Several undesirable effects have been associated with the use of U73122; thus, the specificity of this inhibitor as a PI inhibitor has been questioned. At the concentrations used in our study, U73122 did not induce an increase in basal [Ca2+]i levels. Moreover, the finding that it did not inhibit [Ca2+]i oscillations induced by injection of IP3 reveals that the Ca2+ release mechanisms in these eggs were functional. Furthermore, U73122, at concentrations similar to those used in this study, inhibited the activity of PLC7 isolated from fertilized Xenopus oocytes and blocked fertilization-induced IP3 production and Ca2+ release in these oocytes.
These results suggest that U73122 is inhibiting, at least in part, an active component in SF This conclusion is also supported by reports using sea urchin egg homogenates in which Ca2+ responses to SF were abrogated by preincubation of SF with U73122, although the concentrations of the inhibitor used in those studies were significantly higher than dosages previously reported. Preincubation of sea urchin egg homogenates with up to 400 ^M U73122 failed to show complete inhibition of SF-induced Ca2+ release.
The signal transduction pathway(s) utilized by the sperm or SF to signal [Ca2+]i oscillations in mammalian eggs is not fully elucidated. Our results obtained by injection of SF into mouse eggs and Xenopus oocytes show 1) that incubation of SF or eggs with U73122, an inhibitor of the PI pathway, blocks the generation of [Ca2+]i oscillations, 2) that injection of SF induces a significant increase in [IP3]i, 3) that PLC71, PLC72, and PLC84 and/or its splice variants from the sperm are not likely to be the active component(s) of boar SF, and 4) that injecting adenosphostin A, an IP3R agonist, results in a sensitized CICR mechanism(s). We conclude that SF initiates and sustains [Ca2+]i rises in mammalian eggs by persistently activating the PI pathway.
Adenophostin A Sensitizes CICR
Following fertilization or injection of SF, mammalian eggs become highly sensitized to the injection of CaCl2. This sensitized CICR mechanism is thought to be responsible for the persistence of oscillations in fertilized mammalian eggs. CICR in mammalian eggs appears to be mediated by the IP3R, although the molecular mechanism responsible for it is not known. Having demonstrated that SF stimulates IP3 production, IP3 alone may be the sensitizing stimulus. To test this hypothesis, we evaluated the effects of adenophostin A, an IP3R agonist, on long-term sensitization to CaCl2 injections. Adenophostin A was chosen because of its close structural homology to IP3, its high affinity for the IP3R, and its long half-life. As expected, unfertilized control eggs injected with CaCl2 (2.0 mM) alone showed a minor rise following the injection, and the amplitude of this rise reached a mean peak of 60 ± 41 nM (n = 6; Fig. 6A). No additional [Ca2+] rises were observed.
Some of the smaller size bands recognized by the antibodies in the active fraction, upon further fractionation, were segregated into inactive fractions (data not shown). Similarly, fractionation of SF by hydroxyapatite column followed by Western blotting revealed that active fractions F3 and F4 did not contain immunoreactive PLC71 (Fig. 4D); bovine cumulus cell extracts, which contained full size PLC71, did not exhibit Ca2+ oscillation-inducing activity.
PLCy1, PLCy2, and PLCS4 and/or Its Splice Variants Are Not the Active Components of SF
Because injection of SF triggers IP3 production, it may be that the sperm’s Ca2+ releasing component is a PLC. To investigate if some of the known isoforms of PLC may represent the Ca2+ activating signal of SF, we first fractionated pig testis extracts and pig SF by Superose 12, hydroxy-apatite FPLC, and/or ammonium sulfate precipitation and then tested the Ca2+ releasing activity and the presence of PLCs in these fractions. Injection of unfractionated pig testis extracts (n = 10) into mouse eggs induced [Ca2+]i oscillations that closely resembled those initiated by fertilization or triggered by SF injection (Fig. 4A). Also, among the testis extracts Superose 12 and hydroxyapatite fractions, fraction 4 (Fig. 4B; Superose 12) and fractions 5 and 6 (Fig. 5, A and B; hydroxyapatite) exhibited Ca2+ releasing activity, which were the same fractions that showed Ca2+ releasing activity following Superose 12 and hydroxyapatite fractionation of SF preparations.
Injection of SF Stimulates IP3 Production in Xenopus Oocytes
To determine whether SF injection stimulates IP3 production, we measured the intracellular concentration of IP3 ([IP3]i) in single Xenopus oocytes injected with SF using a biological detector cell combined with capillary electrophoresis. The Xenopus oocyte was chosen because of its large size, thus allowing simultaneous Ca2+ monitoring and cytoplasmic sampling for [IP3]i quantification. First, we determined whether injection of SF was able to induce Ca2+ release in Xenopus oocytes. As shown in Figure 3, approximately 20 sec after SF injection, [Ca2+]i was increased. This Ca2+ increase occurred initially at the SF injection site, at the peripheral region of the oocyte, and then spread across the ooplasm over 15-20 min, and this time course was similar to that reported for normally fertilized Xenopus eggs.