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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 0012-1606/00 $35.00 Copyright © 2000 by Academic Press 238 All rights of reproduction in any form reserved. IP3R Down-regulation in Mouse Eggs 239 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, injec- tion 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 superna- tant 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.
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