Down-Regulation of the Inositol 1,4,5-Trisphosphate Receptor In

Developmental Biology 223, 238–250 (2000) doi:10.1006/dbio.2000.9675, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Down-regulation of the Inositol 1,4,5-Trisphosphateprovided by Elsevier - Publisher Connector Receptor in Mouse Eggs Following Fertilization or Parthenogenetic Activation

Teru Jellerette,* Chang Li He,* Hua Wu,* Jan B. Parys,† and Rafael A. Fissore*,1 *Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003; and †Laboratorium voor Fysiologie, K.U. Leuven, Campus Gasthuisberg O/N, Louvain, Belgium

Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca2؉]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or .injected with several Ca2؉-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting ,Exposure to ethanol or ionomycin, which induce a single [Ca2؉]i rise, failed to signal down-regulation of IP3R-1. However Ca2؉]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or] adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modiﬁes the IP3R without stimulating

production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl2 failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very speciﬁc proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1. © 2000 Academic Press ;Key Words: [Ca2؉]i oscillations; oocytes; mammalian eggs; sperm factor; adenophostin A; thimerosal; strontium chloride proteasome.

INTRODUCTION body, pronuclear formation, DNA synthesis, and ﬁrst mi- totic cleavage (Kline and Kline, 1992; for review see Schultz In eggs of all species studied to date fertilization induces and Kopf, 1995). a large intracellular calcium ([Ca2ϩ]i) rise (Jaffe, 1980; Whi- The signaling pathway responsible for initiating and taker and Patel, 1990). In mammals, this fertilization [Ca2ϩ]i mediating Ca2ϩ release during fertilization remains to be response is characterized by the presence of multiple oscil- fully elucidated. It has been shown, however, that mamma- lations that last for several hours (Miyazaki et al., 1986; for lian eggs express several components of the phosphoinosi- ϩ review see Miyazaki et al., 1993). These [Ca2 ]i rises are tide pathway (Williams et al., 1992, Williams et al., 1996, required to induce egg activation, which consists of a 1998), including phosphoinositidase C (PLC; Dupont et al., sequence of events that includes cortical granule exocyto- 1996; Mehlmann et al., 1998). Activation of PLC has been sis, resumption of meiosis and extrusion of the second polar shown to lead to the production of inositol 1,4,5- trisphosphate (IP3), a Ca2ϩ-releasing second messenger (Ber- 2ϩ 1 ridge, 1993). IP3 mediates Ca release by interacting with To whom correspondence should be addressed at Paige Labora- tories, Department of Veterinary and Animal Sciences, University IP3 receptors (IP3R), which are localized in the endoplasmic of Massachusetts, Amherst, MA 01003. Fax: (413) 545-6326. reticulum and form tetrameric complexes (Patel et al., E-mail: rﬁ[email protected]. 1999). Injection of GTP␥[S], a nonhydrolyzable activator of

G proteins and consequently of PLC, induced repetitive MATERIALS AND METHODS Ca2ϩ responses in eggs of several species, demonstrating that this pathway is functional in mammalian eggs Egg Recovery and Culture (Miyazaki, 1988; Fissore et al., 1995). Furthermore, injection of IP3 has also been shown to induce Ca2ϩ release in Mouse eggs or recently fertilized zygotes were recovered from the oviducts of 8- to 20-week-old CD-1 female mice as previously mammalian eggs (Miyazaki et al., 1988; Schultz and Kopf, described (Wu et al., 1998). Mice were superstimulated with an 1995). injection of 5 IU pregnant mare serum gonadotropin (Sigma Chemi- The three defined isoforms of the IP3R are expressed in cal Co., St. Louis, MO; all reagents from Sigma unless specified) mammalian eggs (Fissore et al., 1999; He et al., 1999), and induced to ovulate 40–48 h later by injection of 5 IU human although the IP3R subtype 1 (IP3R-1) is expressed abun- chorionic gonadotropin (hCG; Sigma). To obtain fertilized zygotes, dantly and in overwhelmingly larger amounts than the females were placed overnight with males following the injection other isoforms (Parrington et al., 1998; He et al., 1999). of hCG. Eggs were collected 14 h post-hCG (phCG) injection into a Also, the IP3R-1 protein is expressed in mammalian eggs in Hepes-buffered solution (TL-Hepes) supplemented with 5% heat- a stage-specific manner, suggesting an important role in treated fetal calf serum (FCS; Gibco, Grand Island, NY). Granulosa fertilization. For instance, fewer than 20 mouse and bovine cells were removed by a 5- to 10-min incubation with bovine testis hyaluronidase and oocytes showing no signs of degeneration and eggs are required to detect the IP3R-1 protein by Western first polar body extrusion were selected for these studies. Eggs were et al., et al., blotting (He 1997; Fissore 1999), and the transferred to 50-␮l drops of KSOM (Specialty Media, Phillipsburg, amounts of IP3R-1 protein increase significantly during NJ), in which they were incubated before and after activation for oocyte maturation (Mehlmann et al., 1996; He et al., 1997). variable periods of time under paraffin oil at 36.5°C in a humidified

This increase in receptor density results in an increased atmosphere containing 7% CO2 in air. IP3R responsiveness during oocyte maturation (Fujiwara et al., 1993; Mehlmann et al., 1994). Furthermore, injection of Microinjection Techniques the blocking IP3R-1 monoclonal antibody 18A10 prior to insemination inhibited, in a dose-dependent manner, Microinjection procedures were carried out as previously de- fertilization-associated Ca2ϩ release and activation in scribed (Wu et al., 1998). Briefly, eggs were placed in a 50-␮l mouse eggs (Miyazaki et al., 1992; Xu et al., 1994). microdrop of TL-Hepes supplemented with 2.5% sucrose and 20% Ca2ϩ release through the IP3R system may be controlled, FCS under paraffin oil and injected using manipulators (Narishige, in addition to several other mechanisms (for review see Medical Systems Corp., Great Neck, NY) mounted on a Nikon Diaphot microscope (Nikon, Inc., Garden City, NY). Injection Missiaen et al., 1996; Taylor et al., 1998; Patel et al., 1999), pipettes were loaded by suction from a 2- to 3-␮l drop containing by regulation of the levels of IP3R-1 protein. Studies in one of the following compounds: 0.5 mM fura-2 dextran (fura-2D; somatic cell lines have shown that IP3R down-regulation Molecular Probes, Eugene, OR), 1 mg/ml protein concentration of follows persistent stimulation of IP3 production induced by boar sperm fractions (SF), or 10 ␮M adenophostin A, a powerful activation of cell surface receptors coupled to PLC (Wojcik- IP3R agonist (courtesy of Dr. K. Tanzawa, Sankyo CO, Tokyo, iewicz et al., 1994, 1995; Bokkala and Joseph, 1997; Sipma Japan). All reagents were diluted in buffer containing 75 mM KCl et al., 1998). This degradation of IP3R, which led to de- and 20 mM Hepes, pH 7.0, and were delivered into the ooplasm by creased cellular responsiveness to IP3, was shown to be pneumatic pressure using a PLI-100 picoinjector (Medical Systems specific since it was not accompanied by general protein Corp.). Injection volumes were approximately of 5–10 pl and this ␮ degradation (Wojcikiewicz and Oberdorf, 1996; Bokkala and resulted in intracellular concentrations of approximately 10 ng/ l for SF (2.5–5 sperm equivalents; Wu et al., 1998) and 100 nM for Joseph, 1997), was associated with IP3 binding to the IP3R adenophostin A. (Zhu et al., 1999), and was mediated by the proteasome, a multiprotein cellular complex involved with degradation of ubiquitinated proteins (Bokkala and Joseph, 1997; Oberdorf SF Preparation et al., 1999). During fertilization, mammalian eggs also Cytosolic SF were prepared from boar semen as described exhibit decreased IP3R responsiveness as they progress to (Swann. 1990; Wu et al., 1998). In brief, semen samples were first the pronuclear stage (Fissore and Robl, 1994; Jones et al., washed twice with TL-Hepes, and the pellet was resuspended in a 1995; Machaty et al., 1997), and this appears to be accom- solution containing 75 mM KCl, 20 mM Hepes, 1 mM EDTA, 10 panied by IP3R-1 down-regulation (Parrington et al., 1998; mM glycerophosphate, 1 mM DTT, 200 ␮M PMSF, 10 ␮g/ml He et al., 1999). However, the mechanism(s) that controls pepstatin, and 10 ␮g/ml leupeptin, pH 7.0. The sperm suspension the demise of IP3R-1 in mammalian eggs is not known. was lysed by sonication (XL2020; Heat Systems, Inc., Farmingdale, Moreover, parthenogenetic activation of mammalian eggs, NY) using a small probe at a setting of 3 for 15–25 min at 4°C. The the use of which has become widespread with the advent of sonicated suspension was spun twice at 10,000g, and the supernatant was collected both times and then centrifuged at 100,000g for cloning techniques, can be induced by several agonists that 2ϩ 45 min at 4°C. The resulting clear supernatant was collected as the stimulate single or multiple Ca rises, but their effects on cytosolic fraction. Ultrafiltration membranes (Centricon-30; Ami- IP3R-1 numbers have not been determined. Thus, we inves- con, Beverly, MA) were used to wash the supernatants (75 mM KCl tigated the signaling mechanism that controls IP3R-1 and 20 mM Hepes, pH 7.0). The extracts were then precipitated by down-regulation in mouse eggs including the possible in- exposure to a saturated solution of ammonium sulfate (50% final volvement of the proteasome pathway. concentration), followed by centrifugation at 10,000g for 15 min at

4°C. The precipitates were collected and stored at Ϫ80°C until the in this case were not performed, since fura-2 has also some affinity time of use. for Sr2ϩ. The presence of intracellular Sr2ϩ will therefore interfere and prevent the obtainment of accurate Ca2ϩ and/or Sr2ϩ intracellular concentrations (Hajnoczky and Thomas, 1997). Parthenogenetic Activation Eggs were measured individually in 35-␮l drops of TL-Hepes Several commonly used parthenogenetic agents were used dur- medium placed on a glass coverslip on the bottom of a plastic ing these studies, including ethanol and ionomycin, which induce culture dish under paraffin oil. Fluorescence ratios were measured a single [Ca2ϩ]i rise (Cuthbertson, 1983; Shiina et al., 1993), and every 6 s and readings were taken for1sateach wavelength. Oocytes were first monitored for 10–120 s to establish baseline others such as adenophostin A, SF, thimerosal, and SrCl2 that 2ϩ evoke [Ca2ϩ]i oscillations (Kline and Kline, 1992; Swann, 1992; Sato [Ca ]i values, after which recordings were stopped for 2–6 min to et al., 1998). The activating compounds were either injected into allow for microinjection or addition of reagents. Recordings were eggs (adenophostin A and SF, see microinjection procedures for then restarted and continued for 10–30 min.

details) or added to the eggs’ culture media (thimerosal, SrCl2). Activation was started in all cases at 16 h phCG and eggs were Inhibitor Preparation evaluated 2 h later by observing second polar body extrusion (18 h phCG) and 5 h posttreatment by evaluation of pronuclear forma- Lactacystin (100 ␮M; Calbiochem, La Jolla, CA), a proteasome tion. After the activation procedure and the assigned incubation inhibitor (Mellgren, 1997; Fenteany and Schreiber, 1998), was used period (1, 2, 4, or 8 h), eggs were collected in 5 ␮l Dulbecco’s to determine if the proteasome was involved in down-regulation of phosphate-buffered solution (DPBS)/polyvinylpyrrolidone (3 mg/ the IP3R-1 by SF. Eggs were incubated in the inhibitor for 30 min ml) and stored at Ϫ80°C. The great majority of eggs collected 8 h prior to injections, and injections of SF were carried out in the posttreatment, except those treated with thimerosal, exhibited presence of the inhibitor. Eggs were then cultured in a new drop of pronuclear formation. KSOM, freshly supplemented with lactacystin, for 2 h. Ethanol activation was carried out by exposing eggs to a 7% ethanol solution in TL-Hepes plus 3 mg/ml BSA for 5 min at 37°C. After the treatment, eggs were washed several times in TL-Hepes Western Blot Technique and cultured for 8 h (24 h phCG). Activation with ionomycin and Equal volumes of crude lysates from 15 or 20 mouse eggs and 6-dimethylaminopurine (DMAP), a kinase inhibitor (Susko-Parrish double-strength sample buffer (Laemmli, 1970) were combined as et al., 1994) was accomplished by incubating eggs with 5 ␮M 2ϩ previously described (He et al., 1997). Samples were boiled for 3 ionomycin for 5 min in Ca free-DPBS plus 3 mg/ml BSA. Eggs min and loaded into 4% SDS–polyacrylamide gels. The separated were then washed in TL-Hepes/1 mg/ml BSA, placed in DMAP/ proteins were transferred onto nitrocellulose membranes (Micron KSOM (2 mM) for 4 h, and cultured for 4 h after being carefully Separation, Westboro, MA) using a Mini Trans Blot Cell (Bio-Rad, washed free of DMAP. Eggs activated with SrCl2 were incubated for 2ϩ Hercules, CA) for2hat4°C. The membranes were first washed in 2or4hinaCa -free M-16-like medium supplemented with 10 PBS and 0.05% Tween (PBS-T) and then blocked in 6% nonfat dry ϩ mM SrCl2. The treated eggs were then washed in TL-Hepes 5% milk in PBS-T for 1 h. After several washes in PBS-T, the mem- FCS and cultured for 2, 4, or 6 h, depending on the experiment. For branes were incubated overnight with a rabbit polyclonal antibody thimerosal activation, eggs were exposed to freshly made solutions raised against a 15 amino acid peptide sequence of the C-terminal ␮ of 200 M thimerosal in KSOM for 30 min. Thimerosal-treated end of the IP3R-1 subtype (Rbt04) diluted to 1:3000 in PBS-T (Parys ϩ eggs were washed free of the reagent using TL-Hepes 5%FCS and et al., 1995). Following several washes, the membranes were incubated for 30 min or 1.5, 3.5, or 7.5 h. incubated for 1 h with a secondary antibody coupled to horseradish peroxidase and diluted 1:3000 in PBS-T. The membranes were Fluorescence Recordings and [Ca2؉]i developed using Western blot chemiluminescence reagents (NEN Determinations Life Science Products, Boston, MA) and exposed for 1–3 min to maximum sensitivity film (Kodak, Fisher Scientific; Springfield, Monitoring of [Ca2ϩ]i levels using fura-2D-loaded eggs was car- NJ). Broad-range prestained SDS–PAGE molecular weight markers ried out as previously described (Wu et al., 1998). UV illumination (Bio-Rad) were run in parallel to estimate the molecular weight of was provided by a 75-W xenon arc lamp and 340- and 380-nm the immunoreactive bands. The intensity of the IP3R-1 bands was excitation wavelengths were utilized. The intensity of the UV light quantified using Adobe PhotoShop (Mountain View, CA) essen- was attenuated 32-fold with neutral density filters, and a photo- tially as described by others (Cameron et al., 1995) and plotted multiplier tube quantified the emitted light after it was passed using Sigma plot software (Jandel Scientific Software, San Rafael, through a 500-nm barrier filter. The fluorescence signal was CA). The mean pixel intensity within a selected set area containing averaged for the whole egg. A modified Phoscan 3.0 software each IP3R-1 band was obtained and the same set area was applied program run on a 486 IBM-compatible system controlled the to all lanes for that particular film. The same set area was also rotation of the filter wheel and shutter apparatus to alternate placed in an area of the film where there were no bands and a wavelengths. Free [Ca2ϩ]i was determined from the 340/380 nm background number was taken. This background number was then

ratio of ﬂuorescence. R min and R max were calculated using 10 ␮M subtracted from all IP3R-1 densities for the ﬁlm under consider- 2ϩ fura-2D in Ca -free DPBS supplemented with 2 mM EDTA (R min) ation. The band from metaphase II (MII) eggs was used as a

or 2 mM CaCl2 (R max) and with 60% sucrose to correct for reference and assigned the value of 1. The intensity of the IP3R-1 intracellular viscosity (Grynkiewicz et al., 1985; Poenie, 1990). The band from eggs after treatment with several different parthenoge- same solutions were also used alone for background subtractions. netic agents was calculated relative to 1 and statistically compared. Ca2ϩ measurements carried out in the presence of extracellular To avoid possible saturation of the quantiﬁcation system and to be

SrCl2 are presented as fluorescence ratios of the 340/380 nm sure that quantification was performed in the linear range, four or excitation wavelengths. Calculations of the [Ca2ϩ]i concentration five exposures of each film were obtained and quantified. Under-

FIG. 1. Western blot analysis and quantification of IP3R-1 immunoreactivity in mouse eggs aged in vitro (A and B) or following in vivo fertilization (C and D). Twenty eggs (e) were used in each lane. UF stands for unfertilized and F for fertilized eggs. MII eggs were always at 16 h post-hCG and the indicated times refer to hours phCG. Data are presented as means Ϯ SEM. Treatments under bars that share a common superscript are not significantly different (P Ͼ 0.05). Values are the means of four Western-blotting experiments, performed on different batches of eggs. exposed and overexposed exposures were discarded. Western blot- IP3 injection (Jones et al., 1995; Jones and Whittingham, ting procedures were repeated at least three times and eggs were 1996). To demonstrate if the reduced responsiveness of collected over several different dates. the IP3R system was due to a decrease in the amount of IP3R-1, mouse eggs were either collected after ovulation Statistical Analysis and aged in vitro or collected soon after fertilization and Statistical comparisons of the intensity of IP3R-1 bands and of cultured in vitro for variable periods of time. Unfertilized the Ca2ϩ parameters were performed using one-way ANOVA, and if eggs were cultured for 10 h (24 h phCG) or 16 h (30 h differences were observed between groups, comparisons between phCG), and zygotes were cultured to the pronuclear stage treatments were achieved by applying the Tukey–Kramer method (24 h phCG), to immediately before first mitosis (30 h using JMP IN software (SAS Institute, Cary, NC). Significance was phCG), or to the two-cell stage (40 h phCG). The presence at P Ͻ 0.05. and amounts of IP3R-1 in these eggs were assessed by Western blotting. As shown in Figs. 1A and 1B, aging of RESULTS eggs in vitro had no significant effect on the amount of IP3R-1. Conversely, fertilization induced marked down- Egg Aging and Fertilization Have Dissimilar regulation of the IP3R-1 by the time of pronuclear forma- Effects on IP3R-1 Down-regulation tion (Figs. 1C and 1D). No significant additional down- Aging of eggs or their fertilization has been shown to regulation of the receptor was observed after this time induce a marked decrease in the eggs’ Ca2ϩ responses to (Figs. 1C and 1D).

FIG. 2. Western blot analysis and quantiﬁcation of IP3R-1 immunoreactivity in mouse eggs activated by exposure to 7% ethanol (Et; A and B) or ionomycin/DMAP (Io/D; C and D). Activation was started at 16 h phCG. Et-24hr-1-c were those eggs that at 24 h phCG exhibited pronuclear formation after ethanol exposure, and Et-24hr-2-c were those that divided into two cells within4hofexposure. All eggs at 24 h phCG (8 h postactivation) for all treatments exhibited pronuclear formation. Twenty eggs (e) were used per lane. Treatments under bars with different superscripts are signiﬁcantly different (P Ͻ 0.05). Values are the means of three Western-blotting experiments, performed on different batches of eggs.

Single [Ca2؉]i Rises Induced by Ethanol and Exposure to these agents was unable to signal IP3R-1 Ionomycin Do Not Down-regulate IP3R-1 degradation (Fig. 2), although they induced high rates of activation (not shown). However, fertilization and injection To understand the mechanism(s) by which IP3R-1 is of SF induced down-regulation of the receptor (Figs. 2C and down-regulated during fertilization, we investigated 2ϩ ϩ 2D). These results suggest that [Ca ]i oscillations may be whether inducing a single [Ca2 ]i rise had an impact on ϩ required to induce fertilization-like IP3R-1 down- IP3R-1 degradation. It is well established that single Ca2 regulation. responses, like those induced by ethanol and ionomycin, trigger high rates of activation and initiation of develop- 2؉ ment in aged oocytes (Cuthbertson, 1983; Shiina et al., [Ca ]i Oscillations Induced by Injection of SF and 1993; Susko-Parrish et al., 1994). In addition, DMAP, a Adenophostin A, but Not by Exposure to SrCl2, kinase inhibitor, was added to test the possibility that in Induce IP3R-1 Degradation the absence of oscillations, additional kinase activity down- To test the notion that multiple [Ca2ϩ]i rises are required regulation may stimulate IP3R-1 degradation. Thus, we for down-regulation of IP3R-1, oscillations were initiated in investigated if mouse eggs exposed to 7% ethanol or 5 ␮M mouse eggs by three different compounds that act on ionomycinϩ2 mM DMAP and collected at the pronuclear different molecular targets of the Ca2ϩ signaling pathway. stage (24 h phCG) exhibited down-regulation of the IP3R-1. Injection of SF, which triggers fertilization-like oscillations

FIG. 3. Western blot analysis and quantification of IP3R-1 immunoreactivity in mouse eggs activated by injection of SF (A and B) or adenophostin A (Ad; C and D). Injections were carried out at 16 h phCG and samples taken within 1, 2, 4, and 8 h postinjection. Treatments under bars with different superscripts are significantly different (P Ͻ 0.05). Values are the means of three Western-blotting experiments, performed on different batches of eggs. by presumably stimulating production of IP3 (Jones et al., (Hecker et al., 1989; Bootman et al., 1992; Missiaen et al., 1998), induced marked down-regulation of the IP3R-1 (Figs. 1992). Thus, we investigated if oscillations initiated by 3A and 3B). The down-regulation of IP3R-1 was persistent, co-incubation of eggs with this compound induced IP3R-1 although significant degradation was seen within 1 h degradation. Thimerosal-mediated Ca2ϩ responses induced postinjection (Figs. 3A and 3B). Injection of adenophostin A, rapid and sustained down-regulation of the receptor (Figs. a nonhydrolyzable agonist of the IP3R, also induced signifi- 4C and 4D). These data suggest that IP3R-1 down- cant down-regulation of the receptor (Figs. 3C and 3D). regulation in mouse eggs may not be exclusively signaled Interestingly, this down-regulation was consistently greater by activation of the phosphoinositidase pathway. than the degradation induced by fertilization (P Ͻ 0.05) or by injection of SF (Figs. 3C and 3D). Finally, exposure of ؉ Patterns of [Ca2 ]i Oscillations Are Agonist- eggs to SrCl for 2 or 4 h failed to induce any changes in the 2 Specific amount of IP3R-1 (Figs. 4A and 4B). Together, these results suggest that down-regulation of IP3R-1 during mouse egg Due to the differential effects on IP3R-1 down-regulation fertilization is not exclusively due to the presence of by the different agonists tested, we examined the Ca2ϩ multiple [Ca2ϩ]i rises, but may be associated with [Ca2ϩ]i responses triggered by each of these agonists. As expected, oscillations initiated by activation of the phosphoinositide injection of SF (n ϭ 4 eggs) and adenophostin A (n ϭ 6 eggs) pathway. induced [Ca2ϩ]i rises similar to those initiated by fertilization, but with higher frequency (P Ͻ 0.05; Figs. 5A and 5B, respectively). On the contrary, eggs exposed to SrCl (n ϭ 5 Thimerosal Induces IP3R-1 Down-regulation 2 eggs) exhibited oscillations with lower frequency and these Thimerosal, a thiol-oxidizing agent, has been shown to rises were different from those initiated by the other ago- induce [Ca2ϩ]i rises without stimulating production of IP3 nists in which the first rise and subsequent rises were very

FIG. 4. Western blot analysis and quantification of IP3R-1 immunoreactivity in mouse eggs activated by SrCl2 (A and B) or thimerosal (Th; C and D). Activation was carried out at 16 h phCG and 15 eggs were used per lane. C stands for control and these eggs were cultured for the same amount of time but were not exposed to SrCl2. Treatments under bars that do not share common superscripts are significantly different (P Ͻ 0.05). Values are the means of five Western-blotting experiments, performed on different batches of eggs.

prolonged (Table 1; P Ͻ 0.05; Fig. 5C). Eggs stimulated with Down-regulation of the IP3R-1 Is Mediated by the thimerosal (n ϭ 9 eggs) showed Ca2ϩ responses with low Proteasome frequency (P Ͻ 0.05). However, the amplitude of thimerosal-induced spikes was comparable to those in- IP3R-1 down-regulation in somatic cells has been shown duced by SF and adenophostin A, although the ﬁrst rise was to be mediated by the proteasome (Bokkala and Joseph, of lower amplitude (Table 1; P Ͻ 0.05). The amplitude of 1997; Oberdorf et al., 1999). The proteasome is also likely

SrCl2-induced rises was not compared to those induced by involved in down-regulation of specific proteins that allow the other agonists because fura-2D, in this study, was fertilized mammalian eggs to exit the MII arrest (Kubiak et calibrated to report intracellular Ca2ϩ levels and it is likely al., 1993; for review see Whitaker, 1996). Thus, we deter- that the observed fluorescence changes represent changes in mined if the degradation of IP3R-1 induced by SF injection the concentrations of both cations (Hajnoczky and Thomas, involved a similar pathway. To accomplish this, eggs were 1997). Despite the different Ca2ϩ profiles, the Ca2ϩ re- preincubated and injected with SF in the presence of lacta- sponses induced by all agonists (thimerosal excluded), ap- cystin, a proteasome inhibitor. Activation was allowed to peared physiological as more than 90% of the eggs were proceed for 2 h, at which time the injected eggs were activated and exhibited pronuclear formation (data not removed and prepared for Western blotting. The 2-h time shown). point was chosen because by 1 h postinjection, significant

FIG. 5. [Ca2ϩ]i oscillation proﬁles in mouse eggs induced by several of the agonists used in this study. Injection of SF (ϳ10 ng/␮l intracellular concentration) induced highly repetitive rises (A) similar to those induced by injection of adenophostin A (B; ϳ100 nM intracellular concentration). SrCl2 induced prolonged rises and of slower frequency (C). Thimerosal, which was incubated with eggs for 30 min, induced repetitive rises (D). The presented Ca2ϩ recordings were carried out in three separate experiments and the termination of the oscillations was due to the termination of the recordings rather than to cessation of the oscillations.

down-regulation of IP3R-1 was already observed (Figs. 3A levels of IP3R-1 similar to those observed during fertiliza- and 3B). Degradation of the receptor in SF-injected eggs tion; (c) that initiation of [Ca2ϩ]i oscillations by exposure to incubated in lactacystin was markedly inhibited (Fig. 6). SrCl2 did not signal IP3R-1 degradation; and (d) that down- Furthermore, the effectiveness of the inhibitor on protea- regulation of IP3R-1 is likely to be mediated by the proteasome activity could also be deduced by the ﬁnding that cell some, since down-regulation was prevented by lactacystin, cycle progression, as assessed by extrusion of the polar a proteasome inhibitor. Together, these data suggest that body, was clearly delayed in SF-injected eggs pretreated and IPR3-1 down-regulation in mouse eggs after fertilization is incubated with the inhibitor (not shown). associated with activation of the phosphoinositide/IP3R system and that persistent IP3 production, induced by the sperm during fertilization or by injection of SF in this study, DISCUSSION may regulate the degradation of the IP3R-1. Mammalian oocytes and eggs closely control the number The results of this study in mouse eggs show (a) that of IP3R-1 before and after fertilization. During oocyte parthenogenetic activation induced by a single [Ca2ϩ]i rise maturation, the increase and redistribution of the IP3R-1 initiated by exposure to ethanol or ionomycin/DMAP does protein is intended to maximize the amount and spatial not induce down-regulation of IP3R-1; (b) that initiation of distribution of Ca2ϩ release following sperm penetration oscillations by injection of SF or adenophostin A, or by (Fujiwara et al., 1993; Mehlmann and Kline, 1994; Shiraishi exposure to thimerosal, evoked a marked decrease in the et al., 1995). However, the role and regulation of the decline

TABLE 1 Characteristics of [Ca2ϩ]i Rises Induced by Several Common Agonists in Mouse Eggs

Amplitude of Amplitude of [Ca2ϩ]i oscillation No. of No. of rises ﬁrst rise third rise Duration of Duration of inducing agonist eggs in 5 min* (nM) (nM) ﬁrst rise (s) third rise (s)

Adenophostin A 6 3.9 Ϯ 1.5a,** 620 Ϯ 30a,b 530 Ϯ 100a 260 Ϯ 65a 70 Ϯ 22a SF 4 5.0 Ϯ 1.0a 870 Ϯ 40a 440 Ϯ 35a 330 Ϯ 90a,b 60 Ϯ 18a b b b SrCl2 51Ϯ 0.0 ND*** ND 740 Ϯ 290 360 Ϯ 70 Thimerosal 9 1 Ϯ 0.4b 330 Ϯ 30b 640 Ϯ 90a 180 Ϯ 44a 70 Ϯ 16a

* The frequency of oscillations was monitored during a 5-min period immediately after the return of the ﬁrst rise to baseline values

(adenophostin A or SF) or after the addition of SrCl2 or thimerosal. All values are means Ϯ standard errors of the mean (SEM). The third rise was chosen arbitrarily to represent any subsequent spike in all treatments. ** Values that do not share a common superscript within columns are signiﬁcantly different (P Ͻ 0.05). *** Not done. See explanation in the text.

of IP3R-1 numbers after fertilization is not fully elucidated, receptor for longer periods of time, may be responsible for despite the fact that this decline may be specific since it is the near total down-regulation of the IP3R-1 observed in not observed in unfertilized aged eggs (Parrington et al., adenophostin A-injected eggs. The finding that thimerosal 1998, and present study) or in eggs activated by exposure to also induces down-regulation of the IP3R-1 suggests that ethanol or ionomycin (present study). These results indi- stimulation of the phosphoinositide pathway may not be cate that IP3R-1 down-regulation is not an effect of egg the only mechanism to signal IP3R-1 degradation in mam- activation per se, but may be more closely associated with malian eggs. Thimerosal, an oxidizing agent that does not the number of [Ca2ϩ]i rises or the mechanism by which the trigger IP3 production, has been shown to increase the oscillations are initiated, both of which were tested in this affinity of IP3R-1 for IP3 (Poitras et al., 1993; Kaplin et al., study. 1994; Vanlingen et al., 1999) and it is possible that by this IP3R-down-regulation studies in somatic cells have mechanism it may induce degradation of the IP3R. Alter- shown that degradation of the receptor requires persistent natively, thimerosal has been demonstrated to oxidize stimulation of PLC-coupled cell-surface receptors, since critical cysteine residues in the receptor, inducing a change activation of these receptors that resulted in brief produc- in the conformational state of the IP3R (Sayers et al., 1993) tion of IP3 was unable to induce receptor degradation and, in this manner, it may induce IP3R-1 down-regulation. (Oberdorf et al., 1999). To produce long-term stimulation in A conformational change has been shown to occur in the our study, mouse eggs were injected with SF. SF has IP3R following binding of IP3, resulting in the opening of previously been shown to induce prolonged [Ca2ϩ]i oscilla- the channel (Mignery and Sudhof, 1990). This structural tions that closely mimic those initiated by fertilization change may also be required for the degradation of the (Swann, 1990; Wu et al., 1997; for review see Swann and receptor (Zhu et al., 1999). Therefore, although thimerosal Lai, 1997). These oscillations are mediated by the IP3R as does not stimulate IP3 production, it may signal IP3R-1 Ca2ϩ responses were suppressed by injection of the IP3R-1 degradation by inducing a similar modification of the recep- blocking monoclonal antibody 18A10 (Oda et al., 1999). In tor. the present study, SF induced a marked and persistent In contrast, SrCl2 failed to induce down-regulation of decline of IP3R-1 similar to that observed following fertili- IP3R-1, despite inducing persistent oscillations. SrCl2 has zation. SF has recently been shown to stimulate production been suggested to induce [Ca2ϩ]i oscillations by sensitizing of IP3 in cell-free extracts from sea urchin eggs (Jones et al., aCa2ϩ-induced Ca2ϩ release mechanism, although the pre- 1998), and thus, it is possible that it may induce IP3R-1 cise mechanism is not known (Cheek et al., 1993). The lack down-regulation by stimulating long-term production of of effect of SrCl2-induced responses on IP3R-1 degradation IP3. is in marked contrast with the high rates of egg activation

Our finding that injections of adenophostin A trigger induced by this agonist. This demonstrates that SrCl2- down-regulation of IP3R-1 supports this hypothesis. Adeno- induced oscillations are capable of signaling the degradation phostin A, a product from Penicillium brevicompactum, is of specific egg proteins whose decline is known to be a full IP3R agonist that is approximately 100-fold more required to exit MII (Whitaker, 1996). This clearly indicates potent than IP3. Moreover, adenophostin A has greater that, in contrast to the situation of other egg proteins, the affinity for the IP3R and is not degraded by the IP3- existence of [Ca2ϩ]i oscillations and the resulting egg acti- metabolizing enzymes (Takahashi et al., 1993). These prop- vation are not sufficient to induce down-regulation of erties, which may allow this agonist to remain bound to the IP3R-1.

the proteasome, as addition of the cysteine protease and proteasome inhibitor N-acetyl-Leu-Leu-norleucinal and lactacystin, a highly speciﬁc inhibitor of the proteasome, both blocked the degradation of the receptor (Wojcikiewicz and Oberdorf, 1996; Bokkala and Joseph, 1997; Oberdorf et al., 1999). Our results in mouse eggs showing that lactacystin blocked down-regulation of IP3R-1 induced by injection of SF is evidence that the proteasome pathway is involved in the decline of IP3R-1 numbers in eggs. Whether IP3 binding to its receptor is the exclusive signal for degradation of the receptor in eggs is not known. Ca2ϩ or protein kinase C (PKC), both of which play a role in activation (Gallicano et al., 1995), may also participate in signaling IP3R-1 degrada-

tion. Our finding that SrCl2 induces oscillations without affecting receptor degradation suggest that neither Ca2ϩ nor Ca2ϩ-dependent PKC is critical or sufficient for IP3R-1 demise in mouse eggs. How the decline in IP3R-1 numbers may affect the frequency and duration of [Ca2ϩ]i oscillations remains to be determined. It is likely, however, that it may be involved in the cessation, or decline in frequency/amplitude, of fertilization/agonist-induced [Ca2ϩ]i oscillations, which is observed as activated eggs progress to the pronuclear stage (Fissore and Robl, 1994; Jones et al., 1995; Parrington et al., 1998). It is important to note that concomitant with these changes in IP3R-1, two critical kinase activities also decline in eggs, those of maturation-promoting factor and mitogen- activated protein kinase (Moos et al., 1995). Thus, it will be necessary to determine whether one of these changes is more important than the other in the regulation/cessation of oscillations or if both contribute equally. The use of eggs/zygotes with different numbers of IP3R-1s but at similar cell cycle stage/kinase levels, which can now be generated using the different agonists reported in this study, FIG. 6. Western blot analysis and quantification of IP3R-1 immu- will allow the discrimination of the effects of IP3R numbers noreactivity in mouse eggs activated by SF in the presence or and cell cycle stage on oscillation patterns in mammalian absence of lactacystin (Lac). Injection of SF was carried out at 16 h eggs. phCG. Eggs were preincubated with the inhibitor (100 ␮M) for 30 In summary, the data presented here show that IP3R-1 min prior to SF injection and were cultured in Lac for 2 h after the injections. Fifteen eggs (e) were used per lane. Treatments under down-regulation in mouse eggs is induced by fertilization bars that do not share common superscripts are significantly and by agonists that persistently stimulate the phosphoino- different (P Ͻ 0.05). Values are the means of three Western- sitide pathway/IP3R system. The data also show that the blotting experiments, performed on different batches of eggs. proteasome pathway is likely to mediate the degradation of the IP3R-1. It will now be important to elucidate the molecular mechanism by which the sperm and SF activate the phosphoinositide pathway that results in down- The conformational change induced by binding of IP3 to regulation of the receptor and how the degradation of the its receptor has been suggested to signal IP3R degradation receptor affects the periodicity and duration of the resultant by enhancing IP3R ubiquitination and, consequently, sig- [Ca2ϩ]i rises. naling degradation by the proteasome (Bokkala and Joseph, 1997; Oberdorf et al., 1999). In somatic cells, it has been shown that persistent stimulation of the phosphoinositide ACKNOWLEDGMENTS pathway results in polyubiquitination of the IP3R-1 (Bokkala and Joseph, 1997; Oberdorf et al., 1999), and We thank Drs. R. T. Duby and T. Ducibella for reading the studies using cells expressing mutant IP3Rs-1, which were manuscript and helpful suggestions and Mr. Jeremy Smyth for unable to bind IP3, showed that these receptors were not many helpful comments and discussions. These experiments were degraded or ubiquitinated (Zhu et al., 1999). These studies supported in part by USDA NRI Competitive Grants 97-2919 and also demonstrated that ubiquitinated IP3Rs are degraded by 99-2371 to R.A.F. J.B.P is a Research Associate from the Fund for

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