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Regular Article |
a Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003
b Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560
c Department of Biochemistry, University of Tokyo, Tokyo 108-8039, Japan
ABSTRACT
Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca2+]i) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca2+]i oscillations, although it did not inhibit Ca2+ release caused by injection of inositol 1,4,5-triphosphate (IP3). The inactive analogue, U73343, had no effect on SF-induced Ca2+ responses. To determine at the single cell level whether SF triggers IP3 production concomitantly with a [Ca2+]i rise, SF was injected into Xenopus oocytes and IP3 concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca2+]i and IP3 production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLC
1, PLC2, or PLC
4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca2+ activity in the extracts. Our results revealed that active fractions do not contain PLC1, PLC2, or PLC4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP3 could be the sensitizing stimulus of the Ca2+-induced Ca2+ release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP3 receptor agonist, showed enhanced Ca2+ responses to CaCl2 injections. Thus, SF, and probably sperm, induces [Ca2+]i rises by persistently stimulating IP3 production, which in turn results in long-lasting sensitization of Ca2+-induced Ca2+ release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.
calcium, developmental biology, fertilization, IVF/ART
INTRODUCTION
During mammalian fertilization, the sperm induces oscillatory changes in the egg's intracellular concentration of free calcium ([Ca2+]i) that are essential for initiating egg activation and subsequent embryo development [1, 2]. The mechanism(s) by which fertilization-associated [Ca2+]i oscillations are initiated remains elusive but may involve the activation of the phosphoinositide (PI) pathway [3]. Stimulation of this pathway causes activation of a phospholipase C (PLC) isoform that, in turn, mediates the production of inositol 1,4,5-triphosphate (IP3). IP3 then induces Ca2+ release from the endoplasmic reticulum (ER). In echinoderm and Xenopus eggs, increased turnover of PI lipids and IP3 production have been detected around the time of fertilization, although not in single cells [4–6], whereas in mammalian eggs determination of IP3 production during fertilization has not been possible because of fertilization asynchrony and the small size of the eggs.
Two mechanisms have been hypothesized by which the sperm may initiate the signaling cascade that culminates in [Ca2+]i oscillations [7]. One hypothesis is that the sperm acts on an egg plasma membrane receptor, suggested to be a G-protein or a tyrosine kinase-coupled receptor, which upon sperm binding activates the PI pathway, leading to Ca2+ release. However, there is no direct evidence for a sperm surface receptor on the plasma membrane of mammalian eggs capable of signaling Ca2+ release [8–10]. The second hypothesis is that after gamete fusion the sperm introduces a cytosolic factor into the egg that triggers [Ca2+]i oscillations by interacting with a presently unknown target(s). Important supporting evidence for this hypothesis comes from the fact that injection of sperm cytosolic factors into eggs can induce [Ca2+]i oscillations similar to those observed during fertilization [11–15] and can initiate parthenogenetic development up to the blastocyst stage [16–18]. Similarly, direct injection of a whole sperm into the cytosol of mammalian eggs, which avoids any contact between the sperm and egg plasma membranes, initiates Ca2+ responses that resemble those observed during fertilization [19, 20]. Although a sperm-derived PLC and several other proteins located in potentially different sperm compartments have been proposed to be the active component(s) of sperm factor (SF), the exact nature and location of SF are still unknown [3, 21–23].
The signal transduction pathway by which injection of SF induces [Ca2+]i oscillations in mammalian eggs has been extensively investigated. Recently, results from several laboratories indicate that, similar to fertilization [24, 25], [Ca2+]i oscillations induced by SF can be blocked either by injection of heparin, a nonspecific competitive inhibitor of the IP3 receptor (IP3R), or by injection of a specific antibody against the IP3R [15, 26]. In addition, SF appears to sensitize Ca2+-induced Ca2+ release (CICR), a mechanism shown to be stimulated in recently fertilized eggs and thought to be mediated by the IP3R [24, 27]. Furthermore, fertilized mammalian eggs exhibit downregulation of the IP3R, and SF signals a comparable degradation of this receptor [28–31]. Collectively, these data demonstrate that the sperm and SF initiate [Ca2+]i oscillations in mammalian eggs by activating a similar signaling pathway that is mediated by the IP3R. In sea urchin egg homogenates, addition of SF induced IP3 production and Ca2+ release, and these responses were blocked by preincubation of SF with U73122, a PLC activation inhibitor, suggesting that SF stimulates the PI pathway [32, 33]. Whether or not SF stimulates this signaling pathway in mammalian eggs has not been determined nor has it been shown that SF injection induces IP3 production in single eggs as opposed to homogenates.
In the present study we investigated whether SF stimulates the PI pathway in mouse eggs by incubating eggs and SF with U73122 prior to injection of SF. We then determined, at the single cell level using Xenopus oocytes, whether injection of SF was able to trigger IP3 production. We then tested whether PLC1, PLC2, and PLC4 and/or its splice variants, which have been identified in sperm/testis and/or eggs [9, 34–37], are the active IP3-producing component(s) in our extracts. We also determined whether increased intracellular concentrations of an IP3R agonist in mouse eggs were sufficient to sensitize CICR, a hallmark of fertilization.
MATERIALS AND METHODS
Egg and Oocyte Recovery
Eggs were obtained from the oviducts of CD-1 female mice (6–12 wk old) superovulated by i.p. injection of 5 IU of eCG (Sigma, St. Louis, MO) followed 48 h later by injection of 5 IU of hCG (Sigma) to induce ovulation. Eggs were recovered 14 h post-hCG into a HEPES-buffered solution (TL-Hepes) [38] supplemented with 10% heat-treated fetal calf serum (FCS; Gibco, Grand Island, NY). Cumulus cells were removed with bovine testes hyaluronidase (Sigma). For this study, eggs with the first polar body and no signs of degeneration were chosen. These eggs were placed in 50-µl drops of KSOM (Specialty Media, Phillipsburg, NJ) under paraffin oil at 36.5°C in a humidified atmosphere containing 7% CO2 until the time of injection.
Stage V and stage VI oocytes were obtained from Xenopus laevis as previously described [39]. Oocytes were cultured in NaCl (96 mM), KCl (2 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), Hepes (5 mM, pH 7.6), and sodium pyruvate (2.5 mM) at 18°C until used in experiments.
Preparation of SF and Testis Extracts
Cytosolic SFs were prepared from boar semen as previously described [40]. After washing, the sperm suspension was sonicated and the lysate was ultracentrifuged. The clear supernatant was concentrated with ultrafiltration membranes (Centriprep-30; Amicon, Beverly, MA) to final concentrations of 20–30 mg/ml protein. These extracts were then mixed for 30 min at 4°C with ammonium sulfate at 50% final saturation, the precipitates were collected by centrifugation (10 000 x g, 15 min, 4°C), and the pellets were stored at -20°C until use. Pellets were resuspended in injection buffer (75 mM KCL and 20 mM Hepes, pH 7.0), washed in the same buffer at least three times to remove all traces of ammonium sulfate, and concentrated with ultrafiltration membranes. Samples were aliquoted and frozen at -80°C.
Small pieces of pig testis were homogenized at 4°C in the same buffer used to prepare SF. The homogenized samples were further lysed by two cycles of freeze-thawing in liquid N2. The suspension was then processed in the same manner as the SF preparation but without sonication or ammonium sulfate precipitation. Samples were aliquoted and frozen at -80°C.
Inhibitor Preparations and Loading Conditions
A stock solution of 1-[6-[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]aimno]hexyl]-1H-pyrrole-2,5-dione (U73122; Calbiochem, La Jolla, CA) was prepared by dissolving the compound in chloroform to a final concentration of 5 mM. The solution was then aliquoted, dried under a stream of N2, and stored at -20°C. On the day of use, U73122 aliquots were reconstituted in dimethylsulfoxide and diluted to working concentrations of 10–30 µM in TL-Hepes supplemented with 5% FCS. Eggs were preincubated in 50-µl drops containing 10–20 µM U73122 under paraffin oil for 15 min at 37°C prior to the injection of SF or IP3, and injection drops were also supplemented with the appropriate concentration of U73122. For some experiments, SF was incubated in 10–30 µM U73122 for 15 min at 4°C prior to injection into eggs. In these experiments, eggs were not exposed to the inhibitor. Dithiothreitol (DTT) was used in reversal experiments either by mixing U73122 with DTT prior to SF addition or by adding DTT following the 15-min incubation of SF in U73122. In all experiments, DTT was used at a final concentration of 10 mM. A 5-mM stock solution of U73343, the inactive analogue, was prepared and used in a manner analogous to U73122.
Microinjection Techniques and [Ca2+]i Monitoring in Mouse Eggs
Microinjection procedures were as previously described [40]. Glass micropipettes were filled by suction from a microdrop containing 0.5 mM fura-2 dextran (fura-2D, dextran 10 kDa; Molecular Probes, Eugene, OR), SF/testis extracts (0.5 or 5 mg/ml protein concentration, respectively), IP3 (500 µM; Molecular Probes), U73122 (30 µM), CaCl2 (2.0 mM), or 10 µM adenophostin A, a potent IP3R agonist (kindly provided by Dr. K. Tanzawa, Sankyo Co., Tokyo, Japan). Solutions were expelled into the egg's cytoplasm by pneumatic pressure (PLI-100 picoinjector; Medical System Corp., Great Neck, NY). The injection volume was approximately 5–10 pl and resulted in a final intracellular concentration of the injected compounds of approximately 1.5%–3% of the concentration in the injection pipette.
Fura-2D fluorescence was monitored as previously described [40]. [Ca2+]i concentrations, Rmin, and Rmax were calculated according to the methods of Grynkiewickz et al. [41] and Poenie [42] and as previously reported [40, 43]; Rmin and Rmax values were 0.12 and 1.3, respectively. [Ca2+]i monitoring of mouse eggs was initiated 30–45 min after injection of fura-2D, which was approximately 15 h post-hCG, and ceased before eggs had reached 22 h post-hCG.
The activity of column fractions was arbitrarily classified as follows: - = absence of activity; + = fractions that initiated a first spike within 10–20 sec after the injection but [Ca2+]i rises did not last for 10 min; ++ = fractions that initiated oscillations that lasted for at least 10 min and showed four or fewer rises per 10 min; +++ = fractions whose Ca2+ responses lasted more than 20 min and four or more rises occurred per 10-min interval; ++++ = fractions that induced one rise per minute in the first 10 min of monitoring. In the last group, the decline in activity was minimal for the first 20 min of monitoring. Assessment of the frequency of [Ca2+]i rises for activity determination was done 5 min postinjection and lasted for 10 min. In those cases in which the first rise lasted for more than 5 min, activity was monitored starting as soon as the baseline was reestablished and continued for 10 min.
IP3 Concentration Determination in Xenopus Oocytes
The concentration of IP3 was measured in small regions of single Xenopus oocytes as described by Luzzi et al. [44, 45] with some modifications. Boar SF or SF buffer alone (50 nl, 30 µg/µl) was microinjected into Xenopus oocytes. Five to 8 min after injection, a small sample of cytoplasm (10 nl) at an average distance of 200 µm from the injection site was aspirated into a capillary tube, and electrophoresis was initiated. The elapsed time between the initiation of cytoplasmic sampling and the onset of electrophoresis was 250 msec. The effluent from the capillary tube was directed onto a single permeabilized detector cell, a baby hamster kidney (BHK 21) cell [45], which had been previously loaded with mag-fura-2, a Ca2+-sensitive fluorophore contained only in the ER. After elution onto the detector cell, the sample containing IP3 opened the IP3R channel, releasing Ca2+ from the ER and increasing the fluorescence of the mag-fura-2. Mag-fura-2 fluorescence was monitored and quantified as previously described [44]. The IP3 concentrations were obtained from a previously constructed calibration curve [44, 45].
Ca2+ Imaging in Xenopus Oocytes
To monitor Ca2+ release in Xenopus oocytes, oocytes were loaded with Ca2+ green-1 dextran (70 kDa) at a final concentration of 20 µM 4–10 h prior to experiments. Oocytes were imaged through a 4x lens using a Nikon EC800 microscope equipped with a PCM 2000 confocal scanner and the software Simple (Compix Inc. Imaging Systems, Cramberry, PA). Images were collected in the nonconfocal mode (no pinhole) with excitation at 488 nm. Emitted light was collected in a 30-nm bandpass centered around 520 nm. Before, during, and after injection of SF, images were collected every 20–50 sec.
Superose 12 and Hydroxyapatite Fast Protein Liquid Chromatography
The SF or pig testis extracts were loaded at 4°C onto a Superose 12 HR 10/30 column and a 5-ml hydroxyapatite column using a fast protein liquid chromatography (FPLC) system. Proteins were eluted, collected, and tested for Ca2+ activity as previously described [40].
Western Blots
Proteins from pig testis extracts and pig SF were diluted in sample buffer [46], separated by 8% SDS-PAGE, and transferred to nitrocellulose membranes (Micron Separation, Westboro, MA) essentially as described for boar SF [40]. The membranes were blocked with 6% milk in 0.1% Tween 20, incubated overnight with dilutions of primary antibodies at 4°C, then washed and incubated for 1 h at 4°C with a secondary horseradish peroxidase-coupled antibody. After several washes, membranes were developed using the a detection system according to the manufacturer's instructions (ECL; Amershan, Arlington Heights, IL). The primary antibodies used to probe the blots were a mixed monoclonal antibody against bovine PLC1 (1:1000; Upstate Biotechnology Inc., Lake Placid, NY), a polyclonal antibody raised against a peptide corresponding to the 20 amino acids at the c-terminal end of human PLC2 (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA), and a polyclonal antibody raised against the C-terminal 157 amino acids of PLC4 (1:500), which recognizes all three splicing variants of this enzyme [37].
RESULTS
U73122 Inhibits SF-Induced [Ca2+]i Oscillations
U73122, a membrane-permeable inhibitor of events leading to PLC activation and/or of PLC itself [47, 48], was used in this study to evaluate whether SF-induced [Ca2+]i oscillations are mediated by activation of the PI pathway. Incubation of mouse eggs in 20 µM U73122 for 15 min completely suppressed the ability of SF to initiate [Ca2+]i oscillations (n = 0/7; Fig. 1B). Incubation of eggs with lower concentrations of the inhibitor was not as effective, and SF-induced Ca2+ responses were blocked in only 1/5 (10 µM) and 7/14 (15 µM) eggs (Fig. 1E), although in the few eggs in which the responses were inhibited the inhibition was complete. Incubation with higher U73122 concentrations (25 µM), although effective in blocking Ca2+ responses, resulted in cell death within a couple of hours after exposure, and this higher concentration was not used in our studies. Injection of SF induced normal [Ca2+]i oscillations in untreated eggs (n = 8/8; Fig. 1A) or into eggs incubated for 15 min in 20 µM U73343 (n = 4/4), the inactive analogue of U73122 (Fig. 1C; P < 0.05). In spite of successful inhibition of SF-induced Ca2+ responses by U73122, several detrimental effects have been reported in cells exposed to this inhibitor, including a rise in basal [Ca2+]i levels and emptying of IP3-sensitive internal Ca2+ stores [49, 50]. Thus, to determine if the inhibition of the Ca2+ responses in our studies was due to emptying of the Ca2+ stores, eggs incubated with 20 µM U73122 were injected with 500 µM IP3 (concentration in the pipette). This concentration of IP3 was chosen because it closely reproduced in frequency and duration the oscillations evoked by injection of 0.5 mg/ml SF. Injection of IP3, which acts directly on the IP3R in the ER, induced [Ca2+]i oscillations in all eggs (Fig. 1D; n = 4/4). These results suggest that the inhibitory effect of U73122 on SF-induced [Ca2+]i oscillations is most likely due to its ability to prevent activation of the PI pathway and subsequent IP3 production.
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To assess whether a component(s) of SF was the target of the inhibitory action of U73122, SF was incubated with the inhibitor prior to injection into eggs. Injection of SF preincubated with 30 µM U73122 abrogated the ability of SF to initiate [Ca2+]i oscillations (n = 5/19; Fig. 2B), whereas injection of untreated SF (n = 4/4; Fig. 2A) or injection of SF preincubated with 30 µM U73343 (n = 4/4; Fig. 2C) induced normal Ca2+ responses (P < 0.05). Incubation of SF with lower concentrations of the inhibitor was much less effective; 0/3 (10 µM) and 4/9 (20 µM) SF-injected eggs showed inhibition of oscillations (Fig. 2H). In these experiments, the mixture of SF + 30 µM U73122 was injected into the eggs and it is possible that the injected inhibitor could be acting on an egg molecule rather than inhibiting a sperm component. To test this possibility, 30 µM U73122 was injected into eggs (approximate intracellular concentration of 0.5–1 µM), followed within 10 min by injection of SF. All eggs treated in this manner exhibited [Ca2+]i oscillations (n = 4/4; Fig. 2D), indicating that U73122 was most likely inhibiting a molecule in SF. In addition, we also determined that injection of 30 µM U73122 alone did not cause Ca2+ release or an increase in the egg's basal [Ca2+]i levels (n = 4/4; Fig. 2E), suggesting that the concentration of the inhibitor used in our studies was not toxic to eggs.
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The inhibitory effects of U73122, whose active site contains an electrophilic maleimide group, have been reported to be lost when exposed to reducing agents [47]. To test whether the effects of U73122 on SF activity were reversible by the same mechanism, SF was preincubated simultaneously for 15 min with 30 µM U73122 and 10 mM DTT and then injected into eggs or SF and U73122 were preincubated first and 15 min later DTT was added to the incubation mixture. In both situations, exposure to DTT reversed the inhibitory effects of U73122 on SF (n = 3/3, Fig. 2F; n = 4/4, Fig. 2G; respectively).
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 [44, 45]. 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 [51]. In contrast, injection of SF buffer alone did not result in the generation of a [Ca2+]i rise (data not shown). Five to 8 min after SF injection, a sample of ooplasm was removed from each oocyte (n = 6) and electrophoresed onto the detector cell for [IP3]i measurement. The SF induced a significant rise in [IP3]i of Xenopus oocytes from 40 nM to 1.3 ± 0.5 µM (range, 0.5–2.5 µM). Addition of SF directly onto the detector cell did not elicit a [Ca2+]i rise, suggesting that the active component of SF is not IP3.
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PLC1, PLC2, and PLC4 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, hydroxyapatite 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) [40]. 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 [40]. These data suggest that the Ca2+ releasing protein(s) in testis extracts has a size and phosphate binding ability similar to that of SF and, most likely, is the same protein(s). Western blots of Superose 12 fraction 4 from testis extracts showed that this fraction did not contain either PLC1 or PLC2 (Fig. 4C). In contrast, testis fractions that contained enhanced amounts of PLC1, PLC2, or both PLC isoforms such as fraction 3 showed no Ca2+ releasing activity (Fig. 4, B and C). 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 PLC1 (Fig. 4D); bovine cumulus cell extracts, which contained full size PLC1, did not exhibit Ca2+ oscillation-inducing activity. PLC2 in SF, whose presence was tested after sequential hydroxyapatite and Superose 12 chromatography, was not detected in fraction F4, which had maximal activity (Fig. 4E). In addition, studies using mouse SF, which contained full size PLC1 and PLC2, demonstrated that the Ca2+ releasing activity of these fractions was unaffected by immunodepletion of both PLC isoforms (data not shown).
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Recently, it has been shown that two splicing variants of PLC4, ALTI and ALTII, are abundantly expressed in rat testis [36, 37]. To determine whether PLC4 and/or its splice variants represent the active component in our fractions, hydroxyapatite fractionation of pig testis extracts was carried out. As shown in Figure 5C, Western blotting of fractions 5–7, which were the active fractions, did not reveal the presence of PLC4 and/or its splice variants, although a strongly reacting band was detected in the unfractionated testis extract and in inactive fraction 2. In addition, ammonium sulfate precipitation of pig SF followed by Western blotting revealed that a band with an approximate molecular mass of 90 kDa, presumably one of the splice variants of PLC4, was segregated into the supernatant, which lacked Ca2+ releasing activity (Fig. 5D). Together, these results suggest that PLC1, PLC2, and PLC4 and/or its splice variants are not likely to be responsible for the Ca2+ releasing activity of SF. The possibility that other PLC isoforms constitute the active component in SF was also investigated. However, because of the cross-reactivity of the commercially available antibodies, it was impossible to discern the contribution of any of these isoforms to the Ca2+ releasing activity of SF.
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 [25, 28]. CICR in mammalian eggs appears to be mediated by the IP3R [43], 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 [52, 53]. 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+]i rises were observed. Injection of mouse eggs with 10 µM adenophostin A induced [Ca2+]i oscillations that subsided after a variable amount of time (data not shown). After the adenophostin A-induced oscillations had significantly decreased in frequency or had ceased altogether, injection of CaCl2 (2.0 mM) caused an immediate rise that reached a mean peak [Ca2+]i of 409 ± 27 nM, which was significantly higher than the rise induced by CaCl2 injection in control eggs (P < 0.05; n = 14). Injection of CaCl2 reinitiated oscillations in some adenophostin A-injected eggs that had stopped oscillating (Fig. 6B; n = 4) or increased the frequency of spikes in currently oscillating eggs (n = 5). Therefore, agonists of the IP3R and in particular increased levels of [IP3]i induced by the sperm and SF may be sufficient to sensitize CICR and perpetuate [Ca2+]i oscillations.
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DISCUSSION
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 PLC1, PLC2, and PLC4 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.
SF Stimulates the PI Pathway to Induce [Ca2+]i Oscillations
Fertilization-induced [Ca2+]i oscillations in mouse eggs have been reported to be blocked, in a dose-dependent manner, by incubation of eggs with the aminosteroid U73122 [7]. In our studies, incubation of mouse eggs with 20 µM U73122 also blocked the oscillations triggered by injection of SF and this concentration was similar to that required to block fertilization-associated oscillations [35]. Similarly, preincubation of sea urchin eggs with 20 µM U73122 completely blocked the sperm-induced [Ca2+]i rise [6]. U73122 (30 µM) was also effective in inhibiting SF-induced [Ca2+]i oscillations in mouse eggs if SF, rather than eggs, was preincubated with the inhibitor prior to its injection. 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 [33], although the concentrations of the inhibitor used in those studies were significantly higher than dosages previously reported [6]. Preincubation of sea urchin egg homogenates with up to 400 µM U73122 failed to show complete inhibition of SF-induced Ca2+ release [33]. These discrepancies in effectiveness and dosages of U73122 in sea urchin egg homogenates may be related to the concentration of SF required to trigger Ca2+ release or to the fact that the inhibitor may be partially inactivated when incubated with the homogenates.
U73122 could be inhibiting multiple steps of the PI pathway. Initially, this inhibitor was thought to be specific for PLC [47]. However, additional studies revealed that U73122 was more effective in blocking the activation of PLC by receptor agonists or by GTPS than activation of PLC by [Ca2+]i increases [54]. Similarly, other studies demonstrated inhibition of G
q subunits by U73122, suggesting that the site of action of this inhibitor may be at the level of G-proteins or at the link between G-proteins and PLCs [48, 55, 56]. Thus, the inhibition of SF-induced oscillations by U73122 implicates the PI pathway; however, the nature of the active molecule(s) in SF cannot be deduced from these studies.
Several undesirable effects have been associated with the use of U73122 [49, 50]; 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 PLC isolated from fertilized Xenopus oocytes and blocked fertilization-induced IP3 production and Ca2+ release in these oocytes [57].
In previous studies in nonmammalian eggs, fertilization was accompanied by an increase in the turnover of polyphosphoinositides and [IP3]i. In these studies, [IP3]i concentrations were determined in groups of asynchronous eggs, so the exact relation between the increased [IP3]i and fertilization could not be determined [4–6]. Recently, using an IP3 mass assay quantification procedure, addition of SF into sea urchin homogenates induced IP3 production [33]. However, cellular homogenates do not always reproduce the events occurring in intact cells. For these reasons, it was important to determine whether [IP3]i increased in intact single cells following microinjection of SF. In the present study, we demonstrated that injection of SF induces a large increase in [IP3]i in single Xenopus oocytes. Further, the increases in [IP3]i induced by SF were similar to those induced by addition of lysophosphatidic acid, an agonist that activates a G-protein-coupled receptor in these oocytes [58]. Thus, the finding that boar SF is capable of initiating normal PI activation in Xenopus oocytes reflects the degree of conservation of the SF active component(s). We did not determine whether SF induces IP3 production in mouse eggs. However, SF-induced activation in mouse eggs has been accompanied by progressive downregulation of the IP3R [30]. Degradation of the IP3R was also observed in fertilized eggs, but it was not detected in eggs activated by agents that did not stimulate the PI pathway [30, 31]. Degradation of the IP3R in somatic cells has been demonstrated to be exclusively associated with IP3 production [59]. Thus, these results suggest that fertilization and SF also persistently stimulate the PI pathway in mammalian eggs.
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 [60], mammalian sperm express PLCß1, PLC1, and PLC2 [34, 35]. In addition, mouse testis expresses high levels of PLC1 mRNA, and this mRNA appears confined to spermatogonia cells [61]. Further, two splicing variants of PLC4, ALTI and ALTII, are abundantly expressed in rat testis [36, 37]. Based on these reports and on additional functional studies [33], it has been proposed that a PLC is the active component of SF [3]. 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 [34]. Our fractionation results confirm and extend these findings by demonstrating that although pig testis extracts and SF contain PLC1, PLC2, and PLC4 and/or its splice variants, these enzymes are present in inactive fractions. The splice variants of PLC4 are the most testis-specific of all the known isoforms of PLC, and the finding that they do not represent the active component of testis extracts suggests that the sperm/SF may trigger IP3 production by delivering a different type of molecule. However, we cannot exclude the possibility that one of the untested PLC isoforms, or a novel undiscovered testicular isoform, may be the active component of SF.
Another possibility is that the sperm/SF may carry an activator of the egg PLCs. Mammalian eggs express PLCß1, PLCß3, PLC1, and PLC2 [34, 35, 62], 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 [63]. For example, [Ca2+]i rises are initiated in mammalian eggs after injection of GTPS or when eggs expressing foreign cell-surface receptors coupled to G-proteins or tyrosine kinase pathways are stimulated with appropriate agonists [34, 64, 65]. Thus, it appears unlikely that SF would induce [Ca2+]i oscillations without involving the egg PLCs.
In echinoderms and ascidians, fertilization and SF (ascidians) appear to activate the egg PLCs; expression of Src homology 2 (SH2) domains of PLC1 inhibited the sperm-induced Ca2+ responses [66–69]. In these eggs, a Src-like tyrosine kinase appears to be required to induce activation of PLC and initiation of Ca2+ release [69–73]. In vertebrates (Xenopus and mouse), the role of eggs PLCs is not as obvious because fertilization-induced Ca2+ responses were not inhibited by competing PLC1 SH2 domains or injection of antibodies against Gq proteins, which are supposed to prevent the activation of PLCß [8, 34, 74]. PLC can be activated by mechanisms other than tyrosine phosphorylation at SH2 domains [75], and in most studies, the anti-Gq antibody was raised against the C-terminal portion of this protein, a domain that does not appear to be important for interaction with PLCs [76]. In Xenopus and mouse eggs, fertilization-induced Ca2+ responses appear sensitive to the addition of tyrosine kinase inhibitors [35, 77], and in Xenopus eggs, Src family kinases seem to activate PLC at fertilization [57, 78]. Whether and how SF may activate a Src-like kinase in mammalian eggs is not known, although in somatic cells an Src kinase was shown to be activated by G-type proteins [79–81], and it has been reported that mammalian sperm express a Gq protein [82]. Thus, the active component(s) of SF may stimulate IP3 production by acting upstream of the egg PLCs, although the precise mechanism remains to be elucidated.
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 [53], 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 nonhydrolysable 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. However, enhanced CICR responses are observed in fertilized eggs between [Ca2+]i rises, suggesting that if IP3 is persistently elevated, it could sensitize the IP3R to Ca2+ to produce [Ca2+]i oscillations. The suggestion that persistently elevated [IP3]i induced by the sperm or by SF may be the mechanism responsible for sensitizing CICR was recently strengthened by the finding that uncaging of IP3 at relatively low amounts for a prolonged period of time in mouse eggs induced fertilization-like oscillations and sensitized the CICR mechanism [83].
Our present findings demonstrate for the first time at the single cell level that pig SF stimulates production of IP3. These results now provide the basis for future work on whether the active component of SF is an unidentified testicular isoform of PLC or an activator of the egg PLCs.
ACKNOWLEDGMENTS
We thank Chang Li He for technical assistance with preparation of SF and testis extracts and Dr. Tom Ducibella for reading the manuscript and for helpful suggestions. We thank other members of the laboratories for discussions and comments on the manuscript.
FOOTNOTES
First decision: 24 October 2000.
1 Supported by grants from the USDA (2371) to R.A.F. and the NIH (GM 57015) to N.L.A. 
2 Correspondence. FAX: 413 545 6326; rfissore{at}vasci.umass.edu
3 These authors contributed equally.
Accepted: December 12, 2000.
Received: September 19, 2000.
REFERENCES
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