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Department of Veterinary and Animal Sciences,5 University of Massachusetts, Amherst, Massachusetts 01003
Laboratorium voor Fysiologie,6 Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Hematech, Inc.,7 Sioux Falls, South Dakota 57106
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70%–80% reduction in the number of intact IP3R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP3 analogue, adenophostin A. Functional Ca2+ release analysis revealed that IP3R-1 is the predominant Ca2+ release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca2+]i oscillations in response to fertilization, expression of PLC
(also known as PLCZ1), and adenophostin A. However, lower concentrations of IP3 and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca2+]i oscillations in this reduced IP3R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP3R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP3R-1-mediated Ca2+ release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca2+]i oscillations at the level of IP3R-1.
calcium, fertilization, gamete biology, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the mechanism by which the sperm initiates these [Ca2+]i transients has not been fully elucidated, evidence supports the concept that shortly after fusion of the gametes, the sperm delivers a soluble factor that activates the phosphoinositide (PI) pathway [9, 10], thereby triggering Ca2+ release and egg activation. This factor, the nature of which is not yet known, has collectively been referred to as sperm factor, and is believed to act either as an activator of an egg phospholipase C (PLC) or is itself a PLC ([11]; for a review see [12]). PLCs catalyze the hydrolysis of phosphatidyl 4,5-bisphosphate (PIP2) into two signaling molecules, IP3 and 1,2-diacyl glycerol (DAG). Whereas DAG plays a role in several signaling pathways, including protein kinase C activation, IP3 is a direct mediator of intracellular Ca2+ release by binding to its receptor, the IP3R, a tetrameric ligand-gated Ca2+ channel located in the membrane of the endoplasmic reticulum, the Ca2+ store of the cell.
To date, three isoforms of IP3R have been identified in mammalian cells. In eggs and ovarian cells, all three isoforms are differentially expressed, with the type-1 representing the most abundant isoform [13–15]. During oocyte maturation, expression of IP3R-1 (also known as ITPR1) is up-regulated and its spatial distribution changes from the perinuclear region of the germinal vesicle to the cortex of the arrested metaphase II (MII) egg [16, 17]. It is believed that this increase in IP3R-1 mass and redistribution contributes to an enhanced IP3R-1 sensitivity to IP3 observed at the MII stage [17–19], which increases the Ca2+-releasing potential of the egg just before ovulation and fertilization.
Following fertilization, the IP3R-1 is progressively degraded, leaving intact approximately 50% of the receptor present at MII [19–21]. In somatic cells, mutational studies have shown that IP3R-1 down-regulation is mediated by IP3 binding, which induces ubiquitination and subsequent degradation by the proteasome [22]. A similar mechanism, therefore, is believed to operate in mammalian eggs [20, 21]. IP3R-1 down-regulation is believed to contribute to the desensitization of the IP3R-1 system (i.e., less Ca2+ release in response to IP3), which occurs after fertilization and becomes very evident by the time zygotes reach the first embryonic interphase [23]. Importantly, during this transition to interphase, the concomitant decrease in the activity of the two M-phase kinases (maturation-promoting factor, MPF; and mitogen-activated protein kinase) may further compromise IP3R-1 sensitivity at this stage [23]. Although a firm functional link has not yet been established between these kinases and IP3R-1 function, sequence analysis reveals the presence of several MPF phosphorylation motifs in IP3R-1 [24]. Therefore, whether IP3R-1 down-regulation is solely responsible for the decreased responsiveness of IP3R-1 as zygotes transition to interphase or, alternately, whether the inactivation of M-phase kinases is partly accountable for the decreased receptor function, has not been fully established.
Evidence for a pivotal role of IP3R-1 during fertilization has been firmly established in the mouse and hamster, as injection of an inhibitory antibody targeted against the C-terminus of the IP3R-1 blocked fertilization-associated Ca2+ release [25, 26] and egg activation in a dose-dependent manner [27]. However, in large domestic species such as the bovine and porcine, the functional role of IP3R-1 during fertilization has remained elusive. For instance, injection of a functional blocking IP3R-1 antibody into bovine eggs inhibited sperm-induced [Ca2+]i oscillations in only one-third of the evaluated eggs [6]. Additionally, injection of heparin, a competitive IP3R antagonist, effectively blocked fertilization-induced Ca2+ release in one study [28], but failed to do so in another report [29]. Furthermore, another Ca2+ release channel, the ryanodine receptor (RyR) has been identified as functional in bovine eggs [30], and as of yet, has not been excluded as a potential candidate as a major Ca2+ release channel in bovine eggs. Therefore, whether IP3R-1 is the sole Ca2+ release channel responsible for generating oscillations during bovine fertilization remains to be determined.
To address the functional significance of IP3R-1 during bovine fertilization and activation, we took advantage of the evidence that persistent IP3 binding is naturally responsible for IP3R-1 degradation [13–15, 20, 21]. Hence, at the onset of oocyte maturation, we injected adenophostin A, a nonhydrolyzable analogue of IP3 that displays a higher affinity for IP3R-1 than IP3 itself [31, 32], and which has been shown to nearly deplete IP3R-1 in mouse eggs [20, 21]. Using this approach, we generated MII eggs that had a 70%–80% reduction in the complement of intact IP3R-1. In these eggs, we then ascertained [Ca2+]i responses to IP3R-1 agonists and to other stimulators of the PI pathway, including fertilization and expression of the novel sperm-specific phospholipase C-zeta (PLC; also known as PLCZ1). We reasoned that this reduced IP3R-1 model would facilitate characterization of IP3R-1 function during bovine egg activation, and would help shed light on the numbers of IP3R-1 required to initiate [Ca2+]i oscillations in mammalian eggs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise noted, all chemicals were purchased from Sigma Chemical Company, St. Louis, MO.
Animal Care and Welfare
Animals used for gamete collections for the present studies were handled according to the National Research Council's Animal Care and Welfare Guidelines, and these procedures were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts.
Animals and Gametes
Cow ovaries were obtained from a local abattoir and germinal vesicle-stage oocytes were isolated from follicles 2–8 mm in diameter. Only those with several layers of compact cumulus cells were selected for experimental use. Following isolation, oocytes were placed into vials in 2 ml of TCM-199 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and 50 µM roscovitine (Calbiochem, San Diego, CA) to maintain meiotic arrest [33]. Oocytes were transported to the laboratory at 38°C in a portable incubator (Mini Tube of America, Verona, WI), and upon arrival, were thoroughly washed in Tyrode lactate (TL)-Hepes buffer solution [34] to remove all traces of roscovitine. Oocytes were then matured in 4-well tissue culture plates (Nunclon; VWR, Chicago, IL) in 300 µl of TCM-199 medium supplemented with 10% FCS, 0.1 U/ml of LH (Sioux Biochemical, Sioux Center, IA), and 1 µg/ml of estradiol for 20 h.
Mouse oocytes and eggs were collected from B6D2F1 female mice between 8 and 12 wk of age. Mice were superovulated by sequential injections of 5 U of eCG followed by 5 U of hCG, as previously described [35]. Germinal vesicle-stage oocytes were collected from the ovaries of female mice 48 h following injection of eCG. Following collection, oocytes were matured in 50-µl drops of Chatot-Ziomek-Bavister medium (CZB) + 5 mg/ml BSA medium under paraffin oil at 36.5°C in a humidified atmosphere containing 6% CO2 for 14 h. Eggs at the MII stage were collected from the oviducts of stimulated females 14 h following administration of hCG into TL-Hepes supplemented with 10% FCS. The cumulus cells were removed by exposure to 900 U hyaluronidase for 3–5 min followed by washing in TL-Hepes. Mouse sperm was obtained from the cauda epididymis of 12- to 16-mo-old CD-1 males and collected into injection buffer (IB; 75 mM KCl and 20 mM Hepes, pH 7.0).
Zona-Free In Vitro Fertilization
At 24 h after the onset of maturation (hpm) bovine MII eggs were removed from maturation medium and placed in 1 ml of TL-Hepes solution containing 900 U hyaluronidase. Eggs were then denuded of cumulus cells by brief (4 min) vortexing. Cumulus-free eggs were then treated with 0.25% protease in TL-Hepes for 5–7 min and gently pipetted to remove the zona pellucida. Zona-free eggs were then cultured for 1 h at 38°C to allow recovery of the plasma membrane. Live-motile, mature bull spermatozoa (kindly donated by Genex Corp., Ithaca, NY) were isolated by centrifugation at 400 x g through a dual-density discontinuous gradient of 45% and 90% Percoll. To induce the acrosome reaction, spermatozoa were treated with 5 µM ionomycin (Calbiochem) in TL-Hepes for 2.5 min [36]. Sperm were then washed twice in the same buffer and were resuspended at a final concentration of 1 x 106/ml in fertilization media (described by Brackett and Oliphant [37]) containing zona-free eggs. Insemination was allowed to take place for approximately 5 h, at which point eggs were removed and washed by gentle pipetting in TL-Hepes, and transferred to a [Ca2+]i imaging dish (see below).
Porcine Sperm Extract Preparation
Cytosolic sperm fractions were prepared from boar semen as previously described [35, 38]. Briefly, semen samples were washed twice with TL-Hepes medium and the sperm pellet was resuspended in a solution containing 75 mM KCl, 20 mM Hepes, 1 mM EDTA, 10 mM glycerophosphate, 1 mM dithiothreitol, 200 µM PMSF, 10 µg/ml pepstatin, and 10 µg/ml leupeptin, pH 7.0. The sperm suspension was sonicated for 25– 35 min at 4°C (XL2020; Heat Systems Inc., Farmingdale, NY), the lysate was spun twice at 10 000 x g, and the supernatants were collected. The resulting supernatant was centrifuged at 100 000 x g for 1 h at 4°C and the clear supernatant was used as the cytosolic fraction. Ultrafiltration membranes (Centriprep 10 and 30 and Centricon 30; Amicon, Beverly, MA) were used to wash the supernatant with IB. Crude sperm extracts were mixed with saturated ammonium sulfate to 50% saturation, the precipitates were collected by centrifugation (10 000 x g, 15 min, 4°C), and the pellets stored at –20°C until use. The pellets were resuspended and washed several times in IB, and concentrated with ultrafiltration membranes. Protein concentrations were determined using the bicinchoninic acid (BCA-1; Sigma) assay for protein quantification.
Microinjection and Intracytoplasmic Sperm Injection
Microinjection techniques were carried out as described earlier [39]. In brief, oocytes and eggs were microinjected using Narishige manipulators (MVI, Avon, MA). Glass micropipettes were filled by suction of a microdrop containing either 0.5 mM fura-2 dextran (fura-2D, dextran 10 kDa; Molecular Probes, Eugene, OR), 1–5 µg/µl porcine sperm extract (pSE), 20–500 µM IP3 (Molecular Probes), 1 µg/ml PLC cRNA, or 10– 40 µM adenophostin A (a generous gift from Dr. K. Tanzawa, Sankyo Co., Tokyo, Japan) and injected into the cytoplasm by pneumatic pressure (PL1-100 picoinjector; Harvard Apparatus, Cambridge, MA). Adenophostins, the most potent and specific agonists of IP3R-1, are derived from the fungus Penicillium brevicompactum [31]. Injection volumes ranged from 5 to 10 pl and 15–20 pl in mouse and bovine eggs, respectively. All injections were carried out at room temperature in TL-Hepes with 5% FCS and 2.5% sucrose. Microinjections were carried out at approximately 10 and 22 h postmaturation for bovine oocytes and eggs, respectively, and at approximately 5 and 16 h postmaturation for mouse oocytes and eggs, respectively. Parthenogenetic bovine zygotes were injected at 8 h postactivation, corresponding to approximately 32 h postmaturation.
Intracytoplasmic sperm injection (ICSI) was carried out as previously described [40, 41] using Narishige manipulators on Nikon microscopes. All manipulations were carried out at room temperature in drops of flushing and holding media (FHM; Specialty Media, Phillipsburg, NJ) under light paraffin oil (Fisher Scientific, Springfield, NJ) at approximately 16 h postmaturation. Sperm were washed in IB and mixed 1-1 with 12% polyvinyl pyrrolidone.
[Ca2+]i Imaging
Bovine and mouse eggs were injected with the fluorescent dye Fura-2 dextran or loaded with 1 µM Fura-2 acetoxymethylester (Fura-2 AM; Molecular Probes) supplemented with 0.02% Pluronic F-127 (Molecular Probes) at room temperature for 20 min. [Ca2+]i imaging was carried out as described earlier [39]. In brief, excitation wavelengths of 340 and 380 nm were applied, and the emitted light was quantified after passing through a 500-nm barrier filter by a photomultiplier tube. Neutral density filters attenuated the intensity of the excitation light, and the fluorescent signal was averaged for the whole egg. Eggs were monitored individually in 40-µl drops of TL-Hepes on a glass coverslip on the bottom of a Petri dish covered with paraffin oil. Fluorescence ratios were taken every 4–20 sec for various time points depending on the experiment and reported as the ratios of 340:380 nm fluorescence. In some experiments several eggs were monitored simultaneously using the software Image 1/FL (Universal Imaging, Downingtown, PA), and images were acquired by using an SIT camera (Dage-MTI, Michigan City, IN) attached to an amplifier (Video Scope International Ltd., Sterling, VA).
Western Blot Technique
For Western blot analysis of the IP3R-1 isoform, equal volumes of crude lysates from five bovine oocytes or eggs and double-strength sample buffer [42] were combined as described earlier [20]. Samples were boiled for 3 min and loaded into 4% SDS-polyacrylamide gels. Broad-range prestained SDS-PAGE molecular weight markers (Bio-Rad, Hercules, CA) were run in parallel to estimate the molecular weight of the immunoreactive bands. The separated proteins were transferred onto nitrocellulose membranes (Micro Separation, Westboro, MA) using a Mini Trans Blot Cell (Bio-Rad) for 2 h at 4°C. The membranes were first washed in PBS and 0.1% Tween (PBS-T) and then blocked in 6% nonfat dry milk in PBS-T for 1 h at 4°C. After several washes in PBS-T, the membranes were incubated overnight at 4°C with a rabbit polyclonal antibody raised against a 15 amino acid peptide sequence of the C-terminal end of the IP3R-1 subtype (Rbt03, [43]) diluted to 1:1000 in PBS-T. Following several washings, the membranes were incubated for 1 h at 4°C with a 1:3000 dilution of a horseradish peroxidase (HRP)-coupled goat-anti-rabbit secondary antibody (Bio-Rad). For time course degradation of the IP3R-1, membranes were developed using Western blot chemiluminescence reagents (NEN Life Sciences Products, Boston, MA) and exposed for 1–3 min to maximum sensitivity film (Eastman Kodak, Rochester, NY). For quantification of IP3R-1 immunoreactivity, membranes were exposed using the Kodak Image Station 440 CF and acquired in the linear phase with Kodak 1D Image Analysis Software version 3.5 (Eastman Kodak). The intensity of the IP3R-1 bands was quantified using Adobe PhotoShop (Mountain View, CA) essentially as described by others [44] and plotted using Microsoft Excel (Redmond, WA). The mean pixel intensity within a selected set area containing the IP3R-1 band was obtained, and the same set area was applied to all lanes for that particular film. The same set area was also placed in an area of the film in which there were no bands, and a background value was obtained and was subtracted from the previously obtained values. The band from MII eggs was used as a reference and assigned the value of 100%.
Western blot analysis of PLC in mature bull spermatozoa was carried out using rabbit polyclonal antisera raised against a 19 amino acid sequence from the C-terminal end of mouse PLC [11], donated by Dr. Ken-Ichi Sato (Kobe University, Japan). Sperm proteins were separated by 10% SDS-PAGE, transferred for 1 h to a polyvinylidene difluoride membrane, and probed with PLC antisera at a 1:1000 dilution followed by secondary labeling with a HRP-conjugated goat-anti-rabbit antibody (Bio-Rad) at a 1:3000 dilution. Blocking and antibody incubations were carried out as described above. Immunoreactivity of PLC was observed using the Kodak Image Station 440CF and Image Analysis Software as described above.
Preparation and Microinjection of PLC Complementary RNA
We used pBluescript containing the full-length coding sequence of murine PLC downstream of a T7 promoter (kindly provided by Dr. Kiyoko Fukami, Tokyo University of Pharmacy and Life Science, Japan). This plasmid was linearized at an EcoRI site just beyond the 3' end of the PLC sequence, and cRNA was transcribed in vitro from the linearized DNA template using the T7 mMESSAGE mMACHINE kit (Ambion; Austin, TX) according to the manufacturer's instructions. This reaction results in incorporation of a 7-methyl guanosine cap at the 5' end of the cRNA. A poly(A) tail of approximately 150 bases was also added to the 3' end of the transcript using the Poly(A) Tailing Kit (Ambion). Capped and tailed cRNAs were purified using the MEGAclear kit (Ambion) and were stored at –80°C in single-use aliquots. Complementary RNA was thawed on ice and then centrifuged for 10 min at 12 000 x g at 4°C and injected at a final pipette concentration of 1.0 µg/µl. After injection of cRNA, eggs were placed in Potassium Simplex Optimized Medium (KSOM [45]) supplemented with half-strength essential and nonessential amino acids (Gibco) and 1 mg/ml BSA for 1–1.5 h at 38°C in 5% CO2 and air to allow for translation and accumulation of PLC protein. Injected eggs were removed from KSOM at various time points for [Ca2+]i measurement.
Artificial Activation of Eggs for Pronuclear-Stage IP3 Injection
At 24 hpm bovine MII eggs were removed from maturation medium and placed in 1 ml of TL-Hepes solution containing 900 U hyaluronidase. Eggs were denuded of cumulus cells by brief (4 min) vortexing and were then washed three times through TL-Hepes and finally held in KSOM medium at 38°C in 5% CO2 and air, until 25 hpm. At 25 hpm, bovine MII eggs were placed into 3 ml of a TL-Hepes solution containing 5 µM ionomycin (Calbiochem) for 4 min. Eggs were then washed three times through TL-Hepes and were placed in KSOM medium (38°C, 5% CO2 in air) supplemented with 2 mM 6-dimethylaminopurine (6-DMAP) for a period of 4 h. Eggs were then washed three times through TL-Hepes at 5 min/wash, and were then returned to KSOM medium for an additional 4 h. At 8 h postactivation, pronuclear (PN)-stage parthenogenetic zygotes were removed from KSOM and placed into a 50-µl drop of TL-Hepes containing 2.5% sucrose for microinjection of IP3 (see Microinjection and Intracytoplasmic Sperm Injection section). After [Ca2+]i measurements, the presence of a pronucleus was determined for each embryo by mounting on a glass slide in a solution of 0.5 mg/ml Hoechst (bis-benzamide, 33342) in 90% glycerol, and visualized under UV light at 200x magnification.
Statistics
Data analysis was conducted using JMP IN statistical analysis software (SAS Institute Inc. Cary, NC). Comparisons of immunoreactive IP3R-1 were performed using one-way analysis of variance and Ca2+ release analysis was performed by the Student t-test, each at a level of 5% significance. Each set of experiments was replicated a minimum of three times, except in Figure 1C, where n = 2, and Figure 2C, where n = 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because bovine oocytes at the germinal vesicle stage contain close to a full complement of mature IP3R-1 (Fig. 1), and because mature IP3R-1 turnover is a rather slow process [46], techniques such as morpholino/antisense or RNA interference were not appropriate for rapidly and specifically reducing the IP3R-1 mass in these cells. Therefore, to achieve IP3R-1 down-regulation in bovine oocytes, we resorted to exploiting the phenomenon of agonist-induced IP3R-1 degradation using the potent IP3 analogue adenophostin A. To establish whether IP3R-1 could be effectively and persistently down-regulated using this approach in maturing bovine oocytes, we injected 40 µM adenophostin A (concentration in the pipette; 1 µM intracellular concentration) 10 h after the onset of oocyte maturation, and at 5-h intervals, monitored IP3R-1 mass by Western blotting until it reached 25 hpm. Uninjected control oocytes were monitored at the same time intervals. Western blot analysis revealed that as early as 5 h postinjection (hpi), maturing oocytes had lost the majority of intact IP3R-1 (Fig. 1A), and that these levels remained low until 25 hpm (15 hpi). To accurately quantify the amount of IP3R-1 degraded by 25 hpm, the time at which all subsequent treatments were performed, an additional series of Western blots was conducted with this time point, but with digital exposures acquired in the linear phase to avoid saturation. Figure 1B demonstrates that by 25 hpm, only 28% ± 6% of intact IP3R-1 remained in MII down-regulated eggs (P < 0.05, n = 4), and similar IP3R-1 degradation (75%–80%) was seen in mouse eggs injected with 10 µM of the same agonist (data not shown). Importantly, down-regulating the receptor did not appear to negatively affect maturation rates, for a similar number of injected and uninjected oocytes progressed to MII stage (59/89; 66% vs. 68/91; 75%; P > 0.05), respectively. Thus, these results demonstrate that adenophostin A can be successfully used to down-regulate a significant amount of IP3R-1 during the maturation of bovine oocytes.
Because higher concentrations of adenophostin A were required to down-regulate IP3R-1 in bovine eggs than in mouse eggs, 40 µM vs. 10 µM, respectively (see our previous work, [15, 20]), we ascertained whether the relative amount of IP3R-1 in bovine eggs was different than that in mouse eggs. To carry out these comparisons, we performed immunoblots using in vivo-matured mouse and in vitro-matured bovine eggs. Quantification analysis revealed that 15 mouse eggs contained 41% ± 8% of the amount of immunoreactive IP3R-1 that was detectable in 5 bovine eggs (n = 2). This suggests that bovine eggs contain 7.3 times the amount of IP3R-1 than mouse eggs in an egg-to-egg comparison. However, based on the fact that the volume of a bovine egg is about three times that of a mouse egg, 900 pl vs. 270 pl, respectively, per unit of ooplasm bovine eggs contain about twice the amount of immunoreactive IP3R-1 than mouse eggs. This demonstrates a high concentration of IP3R-1 in bovine eggs, and supports the contention that this is the primary Ca2+ release channel acting during bovine fertilization. This excess of IP3R-1 could also explain the lack of consistency in studies that attempted to block IP3-induced Ca2+ release in this species [6, 29].
Porcine SE-Induced Ca2+ Release and Activation in IP3R-1 Down-Regulated Eggs
Although fertilization-induced Ca2+ release in bovine eggs is believed to be mediated by IP3R-1 [6, 28, 29], definitive evidence has not yet been clearly forwarded. Because it has been documented that soluble sperm extracts trigger Ca2+ release via the PI pathway [47, see Introduction] and because these extracts have been shown to trigger fertilization-like [Ca2+]i oscillations in bovine eggs [48], we evaluated whether pSE-induced Ca2+ release was modified in bovine downregulated (DR) eggs. Mouse IP3R-1 DR eggs were used as an additional control. As expected, injection of 5 µg/µl pSE (concentration in the pipette), a concentration that has been shown to yield [Ca2+]i oscillations similar to those of sperm [47], triggered fertilization-like [Ca2+]i oscillations that lasted in excess of 40 min in 19 of 23 bovine control eggs (Fig. 2A and Table 1). On the contrary, only 6 out of 41 DR eggs exhibited [Ca2+]i oscillations (Fig. 2, B and C; Table 1; P < 0.05), and most eggs showed only 1–2 rises. Importantly, these results extended to other species. For instance, in mouse eggs, injection of 1 µg/µl pSE triggered high-frequency oscillations (n = 12/16), whereas, as anticipated, only a few DR mouse eggs responded to pSE injections (n = 2/10, Table 1).
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It is well documented that decreased [Ca2+]i responses negatively affect egg activation [49]. Because of the aforementioned effects of IP3R-1 mass on [Ca2+]i oscillations, we sought to determine whether DR eggs were competent to be activated. Therefore, to ascertain whether egg activation was impaired, as judged by PN formation, bovine and mouse eggs were injected with pSE at 20 and 14 hpm, respectively. Bovine eggs were evaluated for a pronucleus the following day (approximately 16 hpi) by staining with Hoescht 33342, and mouse eggs were observed for the presence of a pronucleus that was 6–8 hpi in size by brightfield phase-contrast microscopy. Consistent with the observed Ca2+ profiles, high rates of egg activation were observed in both bovine and mouse control eggs injected with pSE, while very few DR eggs formed a pronucleus (Table 2). These results demonstrate that IP3R-1 plays a crucial role in pSE-induced Ca2+ release and activation in bovine and mouse eggs. These findings also illuminate the requirement for repetitive [Ca2+]i oscillations for the transition into embryonic interphase in mammalian embryos.
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Assessment of [Ca2+]i Store Content in Control MII and IP3R-1 DR Eggs
Due to the lower responsiveness of DR eggs to pSE injection, we sought to determine whether the intracellular Ca2+ content was similar in these eggs and control eggs. To evaluate the size of intracellular Ca2+ stores, we incubated control and DR MII eggs in Ca2+-free medium containing 1 mM EGTA for 10 min, and then exposed the eggs to 5 µM ionomycin. Chelation of extracellular Ca2+ by addition of EGTA allows accurate measurement of the [Ca2+]i pool because it excludes any contribution of extracellular Ca2+ toward the rise. Figure 2C shows representative Ca2+ profiles from each group. The peak [Ca2+]i ratio, F(340/ 380), in control and DR eggs, was similar (0.5 ± 0.08 and 0.6 ± 0.04, respectively; P > 0.05; n = 5). Likewise, no differences were detected in the duration of [Ca2+]i rise between control and DR eggs (6.1 ± 1.7 and 9.0 ± 1.4 min; P > 0.05), respectively. These results demonstrate that the Ca2+ store content of DR and control eggs is similar, and supports the notion that the impaired [Ca2+]i releasing ability of DR eggs induced by pSE is a direct consequence of decreased IP3R-1 mass.
Impact of IP3R-1 Mass on Adenophostin A and IP3-Induced Ca2+ Release in Bovine Eggs
Because our previous results showed that differences in the relative mass of IP3R-1 have a profound effect on pSE-induced Ca2+ release and egg activation, but they do not completely abolish Ca2+ responses, we assessed IP3-induced Ca2+ release (IICR) in these eggs. To accomplish this we injected the aforementioned adenophostin A that has higher affinity for IP3R-1 than IP3 [31, 32]. Injection of 20 µM adenophostin A (concentration in the pipette; 0.5 µM intracellular concentration) into control and DR eggs triggered a series of long-lasting oscillations in all eggs of both groups (n = 5/5), the frequency of which was, surprisingly, indistinguishable during the time monitored, although there was some egg-to-egg variability in the responses (P > 0.05; Fig. 3, A and B). This result suggests that under saturating conditions of IP3, DR eggs with only 20%–30% of intact IP3R-1 are still capable of mounting typical agonist-induced [Ca2+]i responses.
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To ascertain whether the threshold level of IICR in DR eggs differs from that of controls, we chose to test a range of IP3 concentrations from saturating to near-threshold physiological levels. Accordingly, we injected 500 and 20 µM IP3 into control and DR eggs (concentrations in the pipette; 10 µM and 0.5 µM intracellular concentrations, respectively), and measured Ca2+ release parameters. Injection of 500 µM IP3 into control and DR eggs triggered a single [Ca2+]i rise in all eggs examined (Fig. 4 and Table 3). No differences were detected with respect to the amplitude or the duration of the response between control and DR eggs (P > 0.05). However, injection of 20 µM IP3, a 25-fold lower concentration, triggered a [Ca2+]i rise of smaller amplitude in DR eggs compared with that of controls (P < 0.05). Collectively, the above results show that injection of high concentrations of IP3 or adenophostin A is able to overcome IICR desensitization induced by a decrease in the numbers of IP3R-1.
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In Vitro Fertilization or Injection of PLC cRNA Initiate Oscillations in IP3R-1 DR Bovine Eggs
Because adenophostin A has greater affinity for IP3R-1 than the natural agonist [32], we were interested in the consequence of a reduced complement of IP3R-1 on the generation of Ca2+ release by physiological agonists. For this study we chose both in vitro fertilization and injection of PLC cRNA. As a control, ICSI studies were carried out in similarly treated mouse eggs, because in these eggs, fertilization by this method initiates consistent [Ca2+]i oscillations.
Zona pellucida-free bovine eggs were inseminated with acrosome-reacted spermatozoa (see Materials and Methods) and incubated for 3–4 h to allow fertilization to take place. Eggs were loaded with Fura-2 AM (3–7 h postinsemination) and Ca2+ was monitored for 60–200 min. Following [Ca2+]i imaging, monospermic fertilization was confirmed by incubating the zygotes in Hoechst 33342 followed by visualization under UV light at 200x magnification on an inverted microscope. Six out of 6 control fertilized eggs oscillated with intervals similar to those reported earlier [6, 39] (Fig. 5A). Remarkably, fertilized DR eggs also showed [Ca2+]i oscillations in 5 of 5 eggs monitored (Fig. 5B) and displayed intervals similar to those of control eggs. In contrast, DR mouse eggs injected with spermatozoa failed to trigger oscillations (Fig. 5D, n = 7), while control eggs exhibited a normal pattern of oscillation that was characteristic of mouse fertilization (Fig. 5C; n = 7).
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These results imply that a full complement of IP3R-1 is not necessary for fertilization-induced Ca2+ release in bovine eggs, or alternately, they may argue that the IP3R-1 is not the sole channel that underlies Ca2+ release in bovine fertilization. Because fertilization in bovine eggs is asynchronous, we chose to initiate synchronous, long-lasting [Ca2+]i oscillations by directly introducing the molecule believed to represent the sperm's active Ca2+ releasing factor, PLC [11]. To accomplish this we injected cRNA encoding the murine PLC sequence, because the bovine for this purpose was not available. We first verified the presence of PLC in bull spermatozoa to confirm this as a potential physiological agent of IP3 production in bovine zygotes. As shown in Figure 6, a single immunoreactive band of 70 kDa representing PLC was detected in as few as 1.0 x 105 bovine spermatozoa. We then proceeded to inject bovine control and DR eggs with 1 µg/µl PLC cRNA and to incubate injected eggs for 1 h to allow protein translation to take place. At 1, 3.5, 4.5, and 5 hpi, groups of control and DR eggs were monitored for [Ca2+]i oscillations. In control eggs, PLC cRNA triggered fertilization-like oscillations that persisted for at least 6 h (Fig. 6, B and D). The mean oscillation interval during the 1–3.5 hpi time period was 38 ± 10 min, and the frequency increased in a time-dependent manner, probably reflecting accumulation of PLC protein over an extended period of time [11, 50]. Expression of PLC cRNA in DR eggs also triggered fertilization-like oscillations, with a mean interval of 40 ± 4 min during the 1–3.5 hpi time period. However, unlike control eggs, the mean interval between oscillations in DR eggs became progressively longer over time, displaying significant differences from their respective time point controls during the 3.5–4.5 hpi and 5–6 hpi measurement periods (P < 0.05; Fig. 6, C and D). Consistent with these results, both control and DR eggs developed pronuclei at approximately 8 hpi (data not shown). Altogether, these results demonstrate that bovine eggs with less than a full complement of IP3R-1 are still capable of generating an oscillatory Ca2+ response following fertilization or injection of PLC cRNA, one that is sufficient to drive exit from meiosis.
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Pronuclear-Stage Parthenogenetic Zygotes Exhibit Decreased Ca2+ Responsiveness
Calcium ion release is diminished in mouse eggs as they progress toward embryonic interphase [5, 51], and this is believed to be the result of a cell-cycle dependent desensitization of IP3R-1 [23]. Nonetheless, it has not been determined whether a similar type of IP3R-1 regulation takes place in bovine zygotes. Notably, a previous study reported that in fertilized bovine eggs, [Ca2+]i oscillations persist through the first interphase, albeit at lower amplitudes than those observed at M-phase [6]. This result is clearly dissimilar to prior observations in mouse eggs in which [Ca2+]i oscillations cease around the time of PN formation [5]. Therefore, a possible explanation for the lower amplitude [Ca2+]i oscillations observed during interphase in fertilized bovine zygotes [6] could be that, in addition to decreased in IP3R-1 numbers, the IP3R-1 undergoes cell cycle-dependent desensitization. To test this notion, we chose to parthenogentically activate MII bovine eggs using ionomycin and 6-DMAP [52], a procedure that does not induce IP3R-1 degradation (Fig. 7C and [20]). Injection of 20 µM IP3 into control MII eggs and PN-stage zygotes evoked a single [Ca2+]i rise, the amplitude of which was smaller in PN-stage zygotes than in control MII eggs (0.2 ± 0.06, n = 11 vs. 0.5 ± 0.06, n = 4, respectively; P < 0.05; Fig. 7, A and B). No differences were observed in the duration of the induced [Ca2+]i rise. Thus, these results suggest that cell cycle stage has a marked effect on the conductivity of IP3R-1 in bovine zygotes containing a full complement of IP3R-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our present findings show that 1) IP3R-1 is highly enriched in bovine oocytes, containing approximately twice the amount of receptor per unit of ooplasm than mouse eggs; 2) the use of adenophostin A is an effective method for inducing down-regulation of IP3R-1 during oocyte maturation, and that this loss of IP3R-1 mass decreases [Ca2+]i responses to injection of threshold concentrations of IP3R-1 agonists; and 3) bovine eggs are capable of mounting [Ca2+]i oscillations in response to sperm or PLC cRNA even when IP3R-1 numbers have been reduced by as much as 70%–80%. Together, these results highlight the pivotal role of IP3R-1 mediated Ca2+ release in bovine fertilization.
Generation of a DR IP3R-1 Egg Model to Study the Impact of Receptor Mass on Ca2+ Release
To address the role of IP3R-1 in the generation of [Ca2+]i oscillations in bovine eggs, we exploited the phenomenon of agonist-induced degradation of IP3R-1 to generate a model system with reduced numbers of intact IP3R-1. Accordingly, we were able to consistently down-regulate 70%–80% of the total receptor and the resultant MII-arrested eggs were then tested for their ability to mount oscillatory Ca2+ responses when treated with various IP3R agonists (or generators thereof) and sperm. Notably, in spite of depleting the majority of IP3R-1, bovine eggs were still able to initiate oscillatory [Ca2+]i responses. Nonetheless, subtle differences in the levels of responsiveness between agents described herein have led us to uncover some important characteristics of IP3R-1 in bovine eggs.
Of all the IP3R-specific agents tested, substantial differences in Ca2+ release parameters were detected only when either low concentrations of IP3 or physiological concentrations pSE were administered. Conversely, injection of substantially higher concentrations of IP3 or adenophostin A, which has approximately a 10-fold greater affinity for the IP3R-1 than IP3 [32], triggered Ca2+ release patterns that were similar in both control and DR eggs. Consistent with these observations, both in vitro fertilization and expression of PLC triggered seemingly comparable patterns of oscillations regardless of the amount of receptor present (100% vs. 20%–30%). Nevertheless, in DR eggs the interval between PLC cRNA-induced [Ca2+]i rises grew progressively longer during the time of monitoring, suggesting that IP3R-1 mass affects the pattern of oscillations and responsiveness of the system.
Even though the above results pointed to a decreased sensitivity of the IP3R-1 system in DR eggs, this desensitization was initially observed only under threshold concentrations of agonist stimulation. This was clearly illustrated with the pSE injection. For instance, we have previously shown that multiple injections of pSE, corresponding to a single sperm equivalent, are mandatory to induce fertilization-like Ca2+ release and down-regulation of the IP3R-1 in mature bovine eggs [48], implying that single sperm equivalents of pSE are unable to replicate sperm-like production of IP3. Therefore, we propose that injection of pSE into DR eggs triggers diminished [Ca2+]i responses because, in a partially desensitized IP3R-1 system, pSE is unable to generate and sustain the elevated threshold levels of IP3 that are needed to maintain oscillations. On the other hand, in the recurrent presence of high concentrations of IP3 such as those induced by fertilization, expression of PLC, or by injection of adenophostin A, [Ca2+]i oscillations ensue even in the presence of a decreased IP3R-1 mass. This ability of physiological agonists of the IP3R-1 system to initiate [Ca2+]i oscillations in partially desensitized eggs speaks to the resilience of the IP3R-1 system in bovine eggs and underscores its significance for the initiation of development.
Biological Significance of the Overwhelming IP3R-1 Mass in Bovine Eggs
From an evolutionary standpoint, one could ask what the biological significance is for such enhanced IP3R-1 responsiveness, and examine why bovine eggs have such large quantities of IP3R-1. On an egg-per-egg basis, we have shown that a single bovine egg contains approximately seven times the amount of IP3R-1 than a single mouse egg (Fig. 1C), which translates to approximately twice the receptor mass on a volume-to-volume basis. In IP3R-1 DR bovine and mouse eggs, reduction of 75% of total-intact receptors results in entirely different Ca2+ responses when subjected to fertilization. For example, in bovine eggs, 25% of the normal receptor mass seems sufficient for generating normal oscillatory responses, whereas in mouse eggs, Ca2+ release is abolished at these IP3R-1 levels. Thus, one could propose that bovine eggs contain higher amounts of IP3R-1 because during fertilization they support more prolonged [Ca2+]i oscillations than mouse eggs. In support of this view, it has been shown that [Ca2+]i oscillations in fertilized bovine eggs can persist for 22 h postinsemination, ceasing at the 2-cell stage [6], whereas in mouse eggs, oscillations cease at approximately 5 h after fertilization, around the time of PN formation. Nonetheless, the functional significance of the longer duration of oscillations in bovine zygotes is yet to be realized, although, given the recent demonstration that the number of [Ca2+]i rises affects mRNA recruitment and protein translation in mouse zygotes [7], it is logical to envision a similar function in bovine zygotes. Therefore, a positive influence of these oscillations on embryonic developmental competence cannot be discounted.
The enhanced endowment of IP3R-1 in bovine eggs is reciprocated by increased concentrations of the Ca2+ oscillation-inducing factor in bull sperm. Demonstration of the enriched concentration of the oscillating factor in bull sperm was obtained from ICSI studies in mouse eggs in which bull sperm-initiated Ca2+ oscillations exhibited higher frequency and persisted longer than those induced by injection of a mouse sperm [41]. What is more, this may also explain why elimination of more than 50% of IP3R-1 mass in mouse eggs impaired the Ca2+ response to fertilization ([53] and present work, Fig. 5), whereas such an effect was not observed in our study in bovine fertilization, even though a greater depletion of IP3R-1 was accomplished. We therefore could envision that during bovine fertilization, an overwhelming amount of IP3 is produced, and that this overcomes the partial desensitization of the IP3R-1 system induced by IP3R-1 mass. Altogether, these results highlight the significance of the IP3R-1 system in bovine fertilization, and underscore the adaptation that gametes have undergone to create species-specific, developmentally optimized, Ca2+ release patterns. Future studies should address the developmental potential of DR IP3R-1 bovine eggs which would be activated by fewer [Ca2+]i responses.
Impact of Cell-Cycle Stage on the Conductivity of the IP3R-1 in Bovine Zygotes
Recent work has suggested a cell-cycle dependency on the regulation of [Ca2+]i oscillations following fertilization [4, 5, 51]. There are several ways by which cross-talk between the Ca2+ releasing machinery and the cell cycle may be coordinated. For instance, sequestration of the Ca2+ releasing factor during PN formation [5] could reduce the rate of IP3 production by harboring the active factor away from its substrate, thereby decreasing the concentrations of IP3 below threshold levels. In this regard, Larman and coworkers recently demonstrated that PLC-induced [Ca2+]i oscillations are regulated in a cell-cycle dependent manner by the targeting of this molecule to the pronucleus [54]. However, the finding in bovine zygotes showing that oscillations persist through the PN stage [6] demonstrates that this mechanism may not be equally effective in this species. Moreover, the evidence of decreased IP3R-1 conductivity in bovine zygotes at the interphase stage, coupled with the findings that at this stage sperm-initiated oscillations progressively decrease in amplitude [6], suggest that other regulatory mechanisms may be responsible for the cell-cycle modulation of IP3R-1-mediated Ca2+ release in these cells. The evidence that M-phase kinases play a pivotal role in regulating endoplasmic reticulum rearrangements during meiotic and zygotic cell cycle transitions, along with the recent demonstration of their involvement in IP3R-1 phosphorylation in somatic cells [24] and in mouse eggs [23], suggest that these kinases may underlie the cross-talk between IP3R-1 and the cell cycle in bovine eggs. Whether the effects on IP3R-1 function are mainly due to direct IP3R-1 phosphorylation or to redistribution of the receptor remains to be investigated.
In summary, by generating functional IP3R-1 DR bovine eggs, we show that IP3R-1 is the predominant, if not exclusive, Ca2+ release channel responsible for mediating oscillations in these eggs. Furthermore, we demonstrate that changes in the sensitivity of IP3R-1 may contribute to the decreased frequency and amplitude of [Ca2+]i oscillations in bovine zygotes progressing into first mitosis [6]. Last, our DR IP3R-1 egg model will be useful for overexpression studies involving other IP3R subtypes, and for investigating their effects on the spatial and temporal dynamics of [Ca2+]i oscillations.
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ACKNOWLEDGMENTS |
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cDNA, Dr. Ken-Ichi Sato (Kobe University, Japan) for PLC antibody, Dr. K. Tanzawa (Sankyo CO, Tokyo, Japan) for adenophostin A, Genex Corp. (Ithaca, NY) for bull sperm, and Hematech, Inc. (Sioux Falls, SD) for bovine oocytes. We also acknowledge Dr. Manabu Kurokawa and Dr. Jeremy Smyth for lending their expertise in many helpful discussions. |
FOOTNOTES |
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2 Correspondence: Rafael A. Fissore, Department of Veterinary and Animal Sciences, Paige Laboratory, University of Massachusetts, Amherst, MA 01003. FAX: 413 545 6326; rfissore{at}vasci.umass.edu
3 These authors contributed equally to this work
4 Current address: Department of Biology, The University of Pennsylvania, Philadelphia, PA 19104
Received: 22 October 2004.
First decision: 21 November 2004.
Accepted: 21 February 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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