Molecular Human Reproduction, Vol. 8, No. 7, 619-629,
July 2002
© 2002 European Society of Human Reproduction and Embryology
Embryology |
Modifications of the Ca2+ release mechanisms of mouse oocytes by fertilization and by sperm factor
1 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA and 2 Instituto de Ciências Biomédicas de Abel Salazar, Universidade Do Porto, Porto, Portugal
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Abstract |
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A cytosolic factor from sperm (SF) is thought to be responsible for the generation of intracellular calcium oscillations ([Ca2+]i) associated with fertilization in mammalian oocytes. Whether or not mouse oocytes injected with SF exhibit modifications of their Ca2+ release mechanisms similar to those observed in fertilized oocytes is not known and this was investigated here by injecting porcine SF (pSF). First, pSF-activated oocytes injected with CaCl2 showed persistent sensitization of the Ca2+-induced Ca2+ release mechanism, but this sensitization was absent in SrCl2-activated oocytes. Second, pSF-injected oocytes re-initiated oscillations when fused with untreated oocytes, although the Ca2+ responses were short-lived compared to those initiated by fertilization. Likewise, in the presence of colcemid, pSF-initiated oscillations were prolonged but ceased in advance of those in fertilized zygotes. Also, pronuclear envelope breakdown induced by okadaic acid was not associated with Ca2+ release in pSF-generated zygotes, whereas it was observed in fertilized zygotes. Finally, roscovitine, an inhibitor of maturation promoting factor, blocked pSF-induced [Ca2+]i oscillations. Together, these results show that pSF-induced [Ca2+]i responses exhibit properties similar to those triggered by the sperm, although the SF's Ca2+ active component(s) may be less stable or more susceptible to degradation, resulting in shorter modification of the oocyte's Ca2+ release mechanisms.
[Ca2+]i oscillations/cell cycle/mammalian oocytes/oocyte activation/parthenogenesis
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Introduction |
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When ovulated, mammalian oocytes are arrested at the metaphase stage of the second meiotic division (MII). Fertilization, which initiates a series of intracellular calcium ([Ca2+]i) oscillations, releases oocytes from the MII arrest, induces activation, and promotes progression into the first mitotic cell cycle (Whitaker and Patel, 1990
; Kline and Kline, 1992). Events characteristic of oocyte activation include cortical granule exocytosis, meiosis resumption and extrusion of the second polar body, pronuclear formation, and the first mitotic cleavage.
How the sperm signals the initiation of [Ca2+]i oscillations remains to be fully elucidated, although it is likely to involve activation of the phosphoinositide (PI) pathway (Miyazaki et al., 1993; Schultz and Kopf, 1995; Swann and Parrington, 1999). Stimulation of this pathway causes activation of a phospholipase C that leads to the production of inositol 1,4,5-trisphosphate (IP3), a Ca2+ releasing second messenger (Berridge, 1993). IP3 triggers Ca2+ release by binding to its receptor, the IP3 receptor (IP3R), which is localized in the endoplasmic reticulum (ER), the Ca2+ store of the cell. Numerous reports have demonstrated the presence of the molecular components of this signalling pathway in mammalian oocytes (Schultz and Kopf, 1995).
Oocyte activation can also be stimulated parthenogenetically in the absence of the sperm by agents that lead to transient increases in [Ca2+]i (Whittingham, 1980). Parthenogenetic agents such as ethanol or ionomycin cause a monotonic rise in [Ca2+]i, unlike the repetitive oscillations induced by the sperm, and although this single [Ca2+]i rise is adequate to activate aged oocytes, it is not sufficient to activate recently ovulated oocytes (Ozil, 1990; Vitullo and Ozil, 1992). Other commonly used parthenogenetic agents, such as SrCl2 and injection of sperm fractions (SF), induce repetitive [Ca2+]i oscillations and are effective in causing high rates of activation in recently ovulated mouse oocytes (Stice and Robl, 1990; Kline and Kline, 1992; Wu et al., 1998).
Despite the fact that several agents induce Ca2+ release and activation, fertilization-associated [Ca2+]i oscillations exhibit several unique features. For example, sperm-induced [Ca2+]i oscillations are long-lasting and are mediated by the IP3R (Miyazaki et al., 1992). This receptor is sensitized in fertilized oocytes and exhibits an enhanced Ca2+-induced Ca2+ release (CICR) mechanism that allows small and localized [Ca2+]i rises to induce widespread Ca2+ release (Igusa and Miyazaki, 1983; Fissore and Robl, 1994). Furthermore, fertilization is accompanied by down-regulation of the IP3R, which appears to be exclusively associated with [Ca2+]i oscillations induced by production of IP3 (Parrington et al., 1998; He et al., 1999; Brind et al., 2000; Jellerette et al., 2000). Of the above-mentioned activation procedures, only injection of SF is able to closely replicate these changes in the oocyte's Ca2+ release mechanism(s) induced by fertilization. For instance, injection of SF initiates [Ca2+]i oscillations similar to those observed during fertilization in oocytes of all mammalian species studied to date (Swann and Lai, 1997; Wu et al., 1997), and these oscillations evoke oocyte activation and full embryo development (Wu et al., 1998; Sakurai et al., 1999). Similar to fertilization, SF-induced [Ca2+]i oscillations can be blocked either by injection of heparin, a competitive inhibitor of the IP3R, or by injection of a specific antibody against the IP3R (Wu et al., 1997; Oda et al., 1999). Injection of SF also signals down-regulation of the IP3R (Jellerette et al., 2000) and sensitizes CICR (Swann, 1994).
Other presumably exclusive characteristics of fertilized oocytes are the ability to activate oocytes long after fertilization has occurred, and the cell-cycle dependence of [Ca2+]i oscillations. For instance, fusion of fertilized zygotes with MII-arrested oocytes induces activation of the recipient oocytes, but parthenogenetically generated zygotes are unable to do so (Zernicka-Goetz et al., 1995), and it is thought that this property of fertilized zygotes is due to their ability to stimulate oscillations in the arrested oocytes (Zernicka-Goetz et al., 1995). Regarding the cell-cycle dependence, it has been shown that fertilization-associated oscillations last for several hours and cease during entry into interphase, at the time of pronuclear formation (Jones et al., 1995; Day et al., 2000). Whether or not SF-injected oocytes have the capacity to stimulate fertilization-like oscillations in recipient MII oocytes several hours after the initiated oscillations have ceased, and whether they exhibit the modifications in their Ca2+ release mechanism(s) commonly associated with fertilization, is not known and will be examined in this report.
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Materials and methods |
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Oocyte and zygote collection
MII oocytes were collected from the oviducts of CD-1 female mice stimulated with 5 IU of equine chorionic gonadotrophin (Sigma, St Louis, MO, USA) followed 48 h later by 5 IU of HCG (Sigma) to induce ovulation. Fertilized embryos were obtained by mating the females after the injection of HCG. MII oocytes and zygotes were collected 14–15 h post-HCG into a HEPES-buffered solution [Tyrode's lactate (TL)–HEPES] supplemented with 5% heat-treated fetal calf serum (FCS; Gibco, Grand Island, NY, USA). Oocytes were freed from cumulus-cells by a short incubation in bovine testis hyaluronidase (Sigma), and only oocytes that had extruded the first polar body and showed no signs of degeneration were chosen for future experimentation. MII oocytes and zygotes were cultured in 50 µl drops of potassium simplex optimized medium (KSOM; Specialty Media, Lavallette, NJ, USA) under paraffin oil at 36.5°C in a humidified atmosphere containing 5.5% CO2.
Microinjection techniques and parthenogenetic activation
Microinjection procedures were performed according to published techniques (Wu et al., 1997). In brief, oocytes were microinjected in 50 µl drops of TL–HEPES supplemented with 2.5% sucrose (w/v) using a Nikon Diaphot microscope (Nikon, Inc., Garden City, NY, USA) and Narishige manipulators. Glass micropipettes were filled by suction from a microdrop containing porcine sperm factor (pSF) (1.0 µg/µl protein concentration), 2 mmol/l CaCl2 (Sigma), or 0.5 mmol/l Fura-2 dextran (Fura-2 D, dextran 10 kDa; Molecular Probes, Eugene, OR, USA; all concentrations are concentrations in the pipette). A picoinjector (PLI-100; Harvard Apparatus, Cambridge, MA, USA) was used to inject all reagents into the cytoplasm of oocytes by pneumatic pressure. The amount of injected solution was 5–10 pl (Lee, 1989), resulting in final intracellular concentrations of the compounds of 1.5–3% of the concentration in the injection pipette.
ICSI was performed as previously described (Kimura and Yanagimachi, 1995; Fukami et al., 2001). In brief, sperm from 7–11 week old CD-1 males were collected from the tail of the epididymides and washed in buffer that consisted of 100 mmol/l KCl and 10 mmol/l HEPES, pH 7.0. This sperm solution was mixed 1:1 with a 12% solution of polyvinylpyrrolidone and a 5 µl drop was prepared from which a single sperm was aspirated tail-first into a 10 µm blunt-ended pipette driven by a piezo electric unit (Piezo-drill; Burleigh, Rochester, NY, USA). Several piezo pulses were applied to separate the tail from the head, and additional piezo pulses were used to penetrate the zona pellucida and the plasma membrane of the oocyte. The sperm was gently delivered into the oocyte following the application of a short turn on an IM-55-2 Narishige syringe.
Ethanol and SrCl2 were also used to induce oocyte activation according to protocols previously published (Cuthbertson, 1983; Kline and Kline, 1992). Oocytes were exposed for 7 min to a 7% ethanol solution in TL–HEPES + BSA (3 mg/ml), or exposed for 2 h to a 10 mmol/l SrCl2 solution prepared in an M-16-like Ca2+-free medium. After both treatments, oocytes were washed in TL–HEPES + BSA, transferred to KSOM media, and observed for signs of activation. In those experiments in which the effects of roscovitine, a cdk1 and maturation-promoting factor (MPF) inhibitor (Meijer et al., 1997), on pSF-induced [Ca2+]i oscillations were tested, oocytes were cultured in KSOM containing 200 µmol/l roscovitine (Calbiochem, La Jolla, CA, USA) for 1–2 h. Roscovitine was prepared as a 7 mmol/l stock solution in dimethylsulphoxide (DMSO). In these experiments, to maintain the MII stage arrest, 100 ng/ml colcemid (Sigma) was added to the roscovitine-supplemented medium. In experiments designed to assess the ability of pSF to induce long-term oscillations, pSF-injected oocytes were cultured in KSOM containing 100 ng/ml colcemid for 3 h, and then washed and placed in KSOM, and Ca2+ responses were monitored during and after exposure to colcemid.
Okadaic acid (OA; Sigma), a phosphatase inhibitor, was prepared in DMSO and used at final concentrations of 10 µmol/l.
Fluorescence recordings and [Ca2+]i determination
Oocytes and zygotes were injected with the fluorescent dye Fura-2 dextran or loaded with 1 µmol/l Fura-2 AM supplemented with 0.02% pluronic acid for 20 min at room temperature (Molecular Probes) and Ca2+ values were monitored using a Nikon Diaphot microscope fitted for fluorescence measurements as previously described (Wu et al., 1997; Gordo et al., 2000). [Ca2+]i concentrations, Rmin, and Rmax were calculated according to published methods (Grynkiewicz et al., 1985; Wu et al., 1997). Oocytes, zygotes, and cell pairs were individually monitored for [Ca2+]i levels in 50 µl drops of TL–HEPES medium supplemented with BSA (1 mg/ml) placed on a glass coverslip sealed over an opening in the bottom of a cultured dish and covered with mineral oil. For certain experiments, 2–10 oocytes were measured simultaneously using the software Image 1/FL (Universal Imaging, Downington, PA, USA). Images were acquired using an SIT camera (Dage-MTI, Michigan City, IN, USA) coupled to an amplifier (Video Scope International Ltd, Sterling, VA, USA). [Ca2+]i values were not calibrated in this system and therefore are reported as the ratios of 340/380 nm fluorescence. Fluorescence ratios were obtained every 4 s, after 1 s readings at each wavelength.
In experiments examining the effect of roscovitine on [Ca2+]i oscillations, oocytes were first monitored for 2–5 min to establish baseline [Ca2+]i values, after which the recordings were stopped to allow for microinjection of pSF. Recordings were then restarted and continued for the time established for each experiment. [Ca2+]i measurements were also carried out in pronuclear stage pSF-injected or fertilized embryos to determine whether [Ca2+]i rises accompany pronuclear envelope breakdown (PEBD). For these experiments, Ca2+ monitoring started at the time of addition of OA, which induced PEBD within 
1 h, and the monitoring continued for an additional hour after PEBD.
Electrofusion
Recently ovulated oocytes (15 h post HCG), zygotes, pSF-injected oocytes that had extruded the second polar body, and SrCl2-treated MII oocytes were exposed for 5 min to a 0.25% pronase solution in TL–HEPES to remove the zona pellucida. The different experimental cell pairs were then agglutinated in 300 µg/ml phytohaemagglutinin (PHA; Sigma) in DPBS without BSA for 10 min at 37°C. After agglutination, the cell pairs were transferred to a pulsing dish consisting of two stainless steel wire electrodes 0.5 mm apart attached to a tissue culture dish filled with fusion medium containing 0.3 mol/l mannitol, 100 µmol/l MgCl2, 50 µmol/l CaCl2, and 0.01 g/l BSA. An electrical pulse of 0.8 kV/cm for 70 µs was applied using a BTX Electro-Cell Manipulator 200 (BTX, Inc., San Diego, CA, USA). The cell pairs remained in pulsing medium for 1 min and were then placed in 50 µl drops of KSOM media. In most cases, oocyte fusion was observed within 15–20 min after the electrical pulse. The experimental cell pairs utilized were: (i) recently ovulated MII oocytes fused with: (a) pSF-injected oocytes 2–3 h post injection, (b) fertilized embryos without a visible pronucleus, (c) SrCl2-treated MII oocytes 2 h post activation; (ii) pSF-injected oocytes 2–3 h post injection fused with: (a) pSF-injected oocytes 5–6 h post injection and with a visible pronucleus, (b) SrCl2-treated MII oocytes 5–6 h post activation, and with a visible pronucleus.
pSF preparation
pSF was prepared from boar semen as previously described (Swann et al., 1990) with some modifications (Wu et al., 1997, 1998). In brief, semen samples were washed twice with TL–HEPES medium, and the sperm pellet was resuspended in a solution containing 75 mmol/l KCl, 20 mmol/l HEPES, 1 mmol/l EDTA, 10 mmol/l glycerophosphate, 1 mmol/l dithiothreitol, 200 µmol/l PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin, pH 7.0. The resulting suspension was lysed by sonication for 30–35 min at 4°C (XL2020; Heat Systems Inc., Farmingdale, NY, USA). The lysate was then centrifuged twice at 10 000 g, and the supernatants were collected and ultracentrifuged at 100 000 g for 1 h at 4°C. Ultrafiltration membranes (Centricon 30; Amicon, Beverly, MA, USA) were used to wash the supernatants and concentrate these extracts to 60 mg/ml of protein. The crude sperm extracts were then precipitated by exposure to a saturated solution of ammonium sulphate (50% final concentration), centrifuged at 10 000 g for 15 min at 4°C, and the precipitates were collected and stored at –80°C until use. The injected concentration of 1 mg/ml pSF produced an intracellular concentration of 2.5 sperm equivalents (Wu et al., 1998). Protein concentrations were determined using a Sigma protein determination kit (Sigma). Extracts from mouse sperm (mSF) were also prepared and their injection into mouse oocytes resulted in the generation of [Ca2+]i oscillations. Nevertheless, the protein concentrations of mSF required to generate comparable oscillations were at least 10-fold greater than for pSF, and the oscillations still ceased prematurely when compared with those elicited by injection of pSF. Hence, these studies were conducted exclusively with pSF.
In-vitro protein kinase assays
Histone H1 kinase assays were performed as previously described (Fissore et al., 1996). Briefly, groups of five oocytes were lysed by several repeated cycles of freezing and thawing in H1 kinase buffer (80 mmol/l glycerophosphate, 5 mmol/l EGTA, 15 mmol/l MgCl2, 1 mmol/l dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 500 nmol/l cAMP dependent protein kinase inhibitor), and stored at –80°C until use. A volume of 5 µl of a solution containing 0.2 µg/µl histone H1 (type III-S), 0.7 mmol/l ATP, and 50 µCi of [
-32P]ATP (Amersham, Arlington Heights, IL, USA) was added to 5 µl of the crude oocyte lysates. The reaction was allowed to proceed for 30 min at 30°C and was terminated by the addition of 5 µl of double-strength electrophoresis sample buffer (Laemmli, 1970). Samples were boiled for 3 min and loaded into 12% SDS–polyacrylamide gels. Control samples contained all of the components required for the reaction but lacked oocytes. Phosphorylation of histone H1 was visualized by autoradiography using Dupont's Cronex intensifying screens at –80°C (Dupont, Wilmington, DE, USA). Autoradiographs were quantified using Adobe Photoshop (Mountain View, CA, USA) and plotted using SigmaPlot software (Jandel Scientific Software, San Rafael, CA, USA). Multiple gel exposures were used to avoid saturation of the quantification system. Kinase activity was normalized to the MPF kinase activity in MII oocytes, which was arbitrarily given the value of 1. Data were presented as mean ± SEM of three to five separate experiments.
Statistics
The amplitude and frequency of [Ca2+]i oscillations induced by injection of CaCl2 into pSF-injected and SrCl2-activated oocytes are presented as mean ± SEM and compared using one-way analysis of variance (ANOVA). Comparisons between numbers of zygotes that exhibited Ca2+ release at the time of PEBD were carried out using the 
2-test. Densitometric values of the in-vitro kinase assays were compared using ANOVA. The JMP IN software (SAS Institute, Cary, NC, USA) was used to perform all the statistical analyses. In all cases, significance was set at P < 0.05.
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Results |
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pSF, but not SrCl2, induces sensitization of CICR
Injections of CaCl2 into unfertilized oocytes induce non-regenerative Ca2+ responses that fail to initiate [Ca2+]i oscillations, whereas injections of CaCl2 into fertilized or SF-injected oocytes undergoing [Ca2+]i oscillations induce Ca2+ responses of greater amplitude and increased frequency, demonstrating the presence of a sensitized CICR mechanism in the latter groups of oocytes (Igusa and Miyazaki, 1983
; Fissore and Robl, 1994; Swann, 1994) To determine whether pSF could induce long-term sensitization of CICR, that is whether or not pSF-injected oocytes could still respond to CaCl2 long after the oscillations have ceased, CaCl2 was injected into pSF-activated oocytes 2–3 h after the injection of pSF since we have determined that in our conditions pSF-initiated oscillations lasted for 1 h (Figure 1A; n = 10). Injection of CaCl2 caused an immediate rise that reached a mean amplitude of [Ca2+]i of 290 ± 25 nmol/l (n = 7/7), and in 3/7 oocytes oscillations were re-initiated (Figure 1B). On the contrary, injection of CaCl2 into SrCl2-activated oocytes induced a minor rise in 3/7 oocytes, reaching a mean amplitude of 60 ± 32 nmol/l (P < 0.05), and oscillations were not initiated in any of the injected oocytes (Figure 1C).
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Fusion of pSF-injected oocytes with MII oocytes induces short-lived [Ca2+]i oscillations
Fertilized zygotes have been shown to induce oocyte activation upon fusion with MII-arrested oocytes and this was thought to be associated with the capacity to induce Ca2+ release (Zernicka-Goetz et al., 1995
). Thus, we investigated whether or not pSF-injected oocytes can re-initiate [Ca2+]i oscillations upon fusion with MII oocytes. To accomplish this, oocytes injected with 1 µg/µl pSF were fused within 2 h post-injection with MII-arrested oocytes. Ca2+ monitoring was started as soon as fusion was evident, 15 min after the electrical pulse. [Ca2+]i monitoring revealed that 7/10 cell pairs exhibited [Ca2+]i transients that lasted for 10–15 min and exhibited a consistent, progressive decline in amplitude (Figure 2A, B). To compare these responses with those induced by fertilized zygotes, in-vivo-fertilized oocytes 3–5 h post-fertilization, which at this point were not oscillating but still lacked the presence of visible pronuclei, were fused with MII oocytes and Ca2+ responses monitored. In 6/8 oocytes, Ca2+ transients with high frequency that lasted 10–15 min were detected soon after fusion and these rises were similar to those observed following fusion with pSF-injected oocytes (Figure 2C,D). However, three of these oocytes exhibited in addition a [Ca2+]i rise 30–40 min post fusion (Figure 2D), demonstrating that these oocytes were able to re-start fertilization-like oscillations, which have been shown to occur at 40–80 min intervals following pronuclear transfers (Kono et al., 1995). Conversely, SrCl2-activated oocytes were unable to initiate any kind of Ca2+ response when fused with MII oocytes (0/6 oocytes; Figure 2E).
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pSF-induced Ca2+ responses in colcemid-arrested oocytes
To further explore the characteristics of Ca2+ responses induced by physiological concentrations of pSF, MII oocytes were cultured in the presence of the microtubule inhibitor colcemid and the persistence of pSF-induced oscillations was monitored while oocytes were in the presence of the drug as well as immediately after removal from it. It is well known that oocytes fertilized in the presence of microtubule-disrupting agents such as colcemid and nocodazole are unable to exit the MII stage (Jones et al., 1995
; Winston et al., 1995), although the sperm-induced [Ca2+]i oscillations are not altered and persist throughout the presence of the drug (Jones et al., 1995; Day et al., 2000). As shown in Figure 3A, in the absence of colcemid, pSF-injected oocytes exhibited oscillations that ceased at 1 h post injection (n = 7; see also Figure 1A
). As expected, treatment with colcemid did not affect the Ca2+ responses to ethanol that, as shown, did not induce any responses during the monitoring period (Figure 3B). However, in the presence of colcemid, pSF-induced oscillations were significantly extended and were detected in 12/17 oocytes 2–3 h post-injection (Figure 3C and Table I). Nonetheless, after removal of the drug, pSF-injected oocytes showed a single additional response, whereas 8/10 oocytes fertilized by ICSI continued to oscillate for several hours following wash-out of the drug (Figure 3D and Table I).
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PEBD of pSF-injected oocytes is not associated with [Ca2+]i rises
Fertilized zygotes exhibit at least one [Ca2+]i transient at the time of PEBD, whereas this [Ca2+]i transient is not observed in parthenogenetically activated oocytes (Tombes et al., 1992
; Kono et al., 1996; Day et al., 2000) reflecting, possibly, the ability of the sperm's Ca2+ active molecule to associate with nuclear/perinuclear structures. To examine whether pSF-injected oocytes also exhibit this feature, pSF-injected oocytes were monitored for Ca2+ release for a 40 min interval at the expected time of PEBD. None of the 10 oocytes injected with pSF exhibited a single or multiple [Ca2+]i transients at the time of PEBD (data not shown). To more reliably control the time of PEBD in pSF-injected oocytes, PEBD was precociously induced 6 h post-activation by a 2 h exposure to OA. OA, a phosphatase inhibitor, has been shown to induce PEBD in fertilized oocytes within 30–45 min (Moos et al., 1995). All pSF-injected oocytes exposed to OA exhibited PEBD within 30–60 min, although none of the 27 oocytes monitored showed [Ca2+]i transients (Figure 4A, left panel). Likewise, none of the 21 ethanol-activated oocytes showed a Ca2+ response at the time of PEBD (Figure 4A, right panel), although 20/22 oocytes fertilized by ICSI exhibited a Ca2+ response associated with PEBD after OA exposure (Figure 4A, central panel; P < 0.05). To further confirm the inability of pSF to associate with the pronucleus and trigger Ca2+ release at the time of PEBD, the above experiment was performed similarly, although the time of addition of OA was delayed until approximately the time at which PEBD was expected to spontaneously occur at 13–18 h post-activation. As shown in Figure 4B, 0/10 pSF-injected oocytes showed a Ca2+ response at this time point (left panel), whereas 8/10 ICSI-fertilized oocytes did (central panel). As expected, none of the ethanol-treated oocytes showed Ca2+ responses (Figure 4B, right panel).
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Roscovitine inhibits pSF-induced [Ca2+]i oscillations
It is well known that in mouse oocytes, fertilization-associated [Ca2+]i oscillations cease approximately at the time of pronuclear formation (Jones et al., 1995
; Kono et al., 1996; Deng and Shen, 2000). It has therefore been suggested that the ability to initiate and maintain [Ca2+]i oscillations is related to the cell-cycle stage and, more specifically, to the intracellular levels of MPF activity. To determine whether pSF-induced oscillations are subject to a similar cell-cycle regulation, we first fused pSF-injected oocytes 2 h post injection with pronuclear stage zygotes generated either by exposure to SrCl2 or injection of pSF. None of the six pSF-injected oocytes fused to pronuclear stage zygotes exhibited oscillations, suggesting that oscillations are less likely to take place during the pronuclear stage (Figure 5A, B). We then investigated whether pSF-induced [Ca2+]i oscillations are modified if elicited in the presence of roscovitine, a specific MPF/p34/cyclinB kinase inhibitor (Meijer et al., 1997). Prior to injection, oocytes were cultured in KSOM supplemented with roscovitine for 30 min, and the injections were carried out in media supplemented with roscovitine and colcemid to avoid progression into interphase, a transition which we noticed occurs after prolonged culture in roscovitine. Injection of pSF in roscovitine-treated oocytes had no effect on the timing and amplitude of the first [Ca2+]i spike, but subsequent [Ca2+]i oscillations were completely suppressed (n = 6/7; Figure 5C), although pSF was able to induce the expected Ca2+ responses in 9/9 untreated oocytes (Figure 5D). Exposure of oocytes to roscovitine for 1 or 2 h, as expected, significantly decreased MPF activity (Figure 5E). Nevertheless, in colcemid-treated oocytes, the inactivation of MPF activity by roscovitine was transient and by 2 h after incubation in both drugs, MPF activity returned to near peak values (Figure 5E, F).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The results presented in this study show the following: (i) that mouse oocytes injected with pSF exhibit prolonged sensitization of the CICR mechanism; (ii) that pSF-injected oocytes or fertilized zygotes that have ceased to oscillate can re-initiate [Ca2+]i oscillations upon fusion with unfertilized MII oocytes, although in the case of pSF the re-initiated oscillations are short-lived; (iii) that injection of pSF into colcemid-arrested oocytes prolongs the oscillations, but again the oscillations cease prematurely when compared with those initiated by fertilization; (iv) that zygotes generated by injection of pSF do not show Ca2+ transients at PEBD induced by exposure to OA; and (v) that pSF-initiated [Ca2+]i oscillations in MII oocytes are sensitive to the kinase status of the cell. We conclude that [Ca2+]i oscillations induced by injection of pSF exhibit properties that are highly specific for fertilization, although commonly used extraction/preparation procedures may compromise the stability of its Ca2+ active molecule(s).
pSF sensitizes CICR
CICR is highly sensitized in fertilized oocytes and, by virtue of this mechanism, small Ca2+ rises are able to elicit additional Ca2+ release and generate global Ca2+ elevations (Igusa and Miyazaki, 1983). In addition to fertilized oocytes, SF-injected oocytes also exhibit a sensitized CICR mechanism. However, in previous studies, CICR, which was induced by injection of CaCl2, was elicited and tested as oscillations were still occurring (Swann, 1994). In the present study, it was determined whether CICR was still sensitized in oocytes whose pSF-induced oscillations had stopped for at least 30 min. Our results show that the CICR sensitizing stimulus remains present and functional for periods that exceed the detection of oscillations, suggesting that the cessation of oscillations is not caused by the inactivation of this factor. Instead, changes in the kinase status of the oocyte/zygote and degradation of the IP3R-1, changes which have been shown to occur following fertilization (Choi et al., 1991; Parrington et al., 1998; Jellerette et al., 2000), may cause gradual inactivation of the zygote's Ca2+ release mechanisms resulting in the cessation of oscillations. With regard to the nature of the CICR sensitizing stimulus, which is presently unknown, it has been recently noted that CICR is enhanced in oocytes in which the levels of intracellular IP3 are persistently elevated by slow release of caged IP3 (Jones and Nixon, 2000), or by a bolus injection of adenophostin A, an IP3R agonist (Wu et al., 2001). Further, pSF has been shown to induce production of IP3 in sea urchin oocyte extracts (Jones et al., 1998), and most recently in Xenopus oocytes (Wu et al., 2001). Thus, it seems that the sperm and SF may induce long-term [Ca2+]i oscillations by stimulating persistent production of IP3.
pSF-injected oocytes and re-initiation/persistence of [Ca2+]i oscillations
A feature of fertilized mouse zygotes that is unmatched by zygotes generated by any other parthenogenetic treatment is the ability of fertilized zygotes to induce activation upon fusion with unfertilized MII oocytes (Zernicka-Goetz et al., 1995). This activating capacity appears only to be present until the early 2-cell stage, and it is thought to rely on the ability to re-start Ca2+ release upon fusion (Zernicka-Goetz et al., 1995). The fact that fertilized oocytes have the capacity to re-start Ca2+ release long after oscillations have ceased was first demonstrated by the property of transferred pronuclei to re-start fertilization-like oscillations upon fusion with MII oocytes (Kono et al., 1995). Furthermore, the finding that fertilization in the presence of colcemid results in oscillations that can last for >24 h provided conclusive proof that the sperm's Ca2+ active molecule(s) can support long-term oscillations (Jones et al., 1995; Day et al., 2000). However, whether or not oocytes injected with physiological doses of pSF have the ability to re-start/maintain activity for such prolonged periods has not been explored. The present results show that pSF-injected oocytes are able to induce short-lived Ca2+ responses upon fusion with MII oocytes, while SrCl2-activated zygotes failed to do so. Importantly, however, pSF-injected oocytes are unable to establish fertilization like-oscillations following fusion, whereas fertilized oocytes are able to do so. Likewise, the Ca2+ responses induced by pSF are prolonged in the presence of colcemid but they cease soon after removal of the drug, whereas sperm-induced oscillations persist for several additional hours.
Our present results are, therefore, in disagreement with findings from a previous study that showed that transfer of pronuclei generated by injection of pSF into fresh MII oocytes was able to re-initiate [Ca2+]i oscillations and induce oocyte activation (Kono et al., 1995). Several reasons may account for the differences between the two studies, although none probably as important as the fact that in the earlier study the dose of SF was 
10-fold greater than the dose of pSF used in the present study. We speculate that although the 1–5 sperm equivalents used in our experiments managed to initiate high frequency [Ca2+]i oscillations, they might have been completely degraded/inactivated several hours after the injection. Conversely, in the previous study, sufficient amounts of SF were injected to avoid full degradation and achieve stabilization and protection by association with the pronuclear membrane. Evidence that nuclear membranes may stabilize the factor, and perhaps regulate its release into the cytosol, is suggested by findings that prior to fertilization, the active sperm factor is thought to be localized/associated with the sperm's perinuclear material, the theca (Kimura et al., 1998; Perry et al., 1999). Collectively, our results support a critical role for SF in the initiation of oscillations, but the data suggest that the stability/activity of the active molecule(s) may be compromised. These data also imply that the process of release and solublization of the Ca2+ active molecule(s) from the sperm soon after fertilization must be carefully orchestrated to allow for prolonged, and re-inducible, Ca2+ releasing activity.
We cannot discount the possibility that the reduced duration of oscillations following injection of pSF reported here could be due to accelerated degradation/inactivation of the molecule because of its heterologous origin. Also, we could be injecting lesser amounts of active pSF than the quantity expected to be released by a sperm; the total amount of SF activity present in a sperm is not known. Nonetheless, these reasons are unlikely to explain our results because, as previously mentioned, mouse SF appears significantly less active than pSF, and because the high frequency of the pSF-initiated oscillations demonstrates that the injected extract was highly active. Finally, although the amount of pSF delivered by injection in this study was likely to have been less than the activity present in a porcine sperm, it was probably at least equal to, and most likely greater than, the amount present in a single mouse sperm since our preliminary observations show that porcine sperm contain significantly greater amounts of Ca2+-inducing activity than mouse sperm.
pSF does not induce Ca2+ release at PEBD
Seminal studies in sea urchin embryos have demonstrated that distinct [Ca2+]i rises occur during the first cell cycle and correlate with specific events during progression of the cell cycle (Steinhardt, 1990; Whitaker and Patel, 1990). Similarly, in mammalian zygotes, it has been reported that a [Ca2+]i rise, which in one study was followed by oscillations, accompanies PEBD (Tombes et al., 1992; Jones et al., 1995; Kono et al., 1996). This late Ca2+ release has not been observed in parthenogenetically activated oocytes (Kono et al., 1996). Importantly, additional investigations have revealed that PEBD is associated mostly with a single rise without subsequent oscillations (Day et al., 2000; Tang et al., 2000), that the PEBD-associated Ca2+ release is detected in <50% of the fertilized oocytes, and that it was of very small amplitude (Day et al., 2000). In the present study we evaluated whether or not oocytes activated with physiological concentrations of pSF exhibited Ca2+ release at PEBD. Our results show that pSF-injected oocytes treated with OA failed to show Ca2+ responses prior to or during PEBD, whereas almost all fertilized oocytes did. Therefore, these results confirm and extend our previous findings that a bolus injection of physiological amounts of semi-purified pSF preparations induces limited persistence of Ca2+-releasing activity.
Roscovitine inhibits pSF-induced [Ca2+]i oscillations
Fertilization-induced [Ca2+]i oscillations in mouse oocytes have been suggested to be cell-cycle dependent (Jones et al., 1995); oscillations start at MII soon after fertilization, continue for several hours and terminate just before pronuclear formation, the stage at which the levels of MPF and mitogen-activated protein kinase (MAPK) have decreased (Jones et al., 1995; Deguchi et al., 2000). This possible association between kinases and Ca2+ responsiveness has been further tested in ascidian oocytes and those studies showed that sperm- and SF-triggered [Ca2+]i oscillations are closely associated with MPF activity and less with MAPK levels, since oscillations were maintained in conditions where MPF activity was high and MAPK activity remained at basal levels (McDougall and Levasseur, 1998; Levasseur and McDougall, 2000). Similarly, in mouse oocytes, it has been demonstrated that inactivation of MPF by roscovitine, a specific inhibitor of p34cdc2/cyclin B, abruptly suppressed fertilization-induced [Ca2+]i oscillations (Deng and Shen, 2000). In the present study, we observed that pSF-induced [Ca2+]i oscillations also appear to be influenced by the stage of the cell cycle and MPF levels. For example, pSF-injected oocytes failed to re-initiate oscillations if fused with pronuclear stage embryos and injection of pSF into oocytes pre-incubated with roscovitine blocked the initiation of oscillations.
Nevertheless, it does not seem that MPF alone is exclusively responsible for regulating Ca2+ signalling during the first cell cycle in mammals. For instance, spontaneous, long-lasting [Ca2+]i oscillations are observed at the GV stage in mouse oocytes (Deng et al., 1998), a stage at which MPF is inactive (Choi et al., 1991; Verlhac et al., 1994). Furthermore, fertilization and pSF injection have been shown to induce long-lasting [Ca2+]i oscillations at this stage (Mehlmann and Kline, 1994; Wu et al., 1997; Deng et al., 1998). In addition, studies in bovine, human and rabbit oocytes have shown that fertilization-induced [Ca2+]i oscillations in these species continue throughout the pronuclear stage (Fissore and Robl, 1994; Nakada et al., 1995; Sousa et al., 1996). Finally, although in mouse oocytes Ca2+-releasing activity appears to be enhanced after PEBD, a stage at which MPF activity is high but MAPK activity remains at basal levels, the amplitude and frequency of the oscillations are very different to those initiated at MII (Jones et al., 1995; Day et al., 2000; Tang et al., 2000). Collectively, these findings indicate that a more complex mechanism, probably regulated by changes in the activity of several kinases as well as by the sensitivity of the IP3R and the content of the Ca2+ stores may be involved in controlling Ca2+ responsiveness in mammalian oocytes. In fact, the necessary concentration of roscovitine used in this study to suppress the pSF-induced [Ca2+]i oscillations, 100 µmol/l, was 10-fold higher than that used for arresting the cell cycle in sea urchin and starfish embryos (Meijer et al., 1997) and, at these concentrations, roscovitine may be affecting the oocyte's Ca2+-releasing activity by modifying other presently unidentified kinases/events.
Conclusion
In summary, our present findings demonstrate that [Ca2+]i oscillations initiated by pSF exhibit properties very similar to those triggered by the sperm, and thus the pSF's Ca2+ active molecule may represent the sperm's active molecule(s). Nevertheless, our results also show that the SF's Ca2+ active component(s) may be less stable or more susceptible to degradation, possibly due to current methods of preparation. This lack of stability may explain, at least in part, the inability thus far to isolate the SF's active compound(s) via biochemical approaches.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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This work was supported in part by a grant from the USDA (99–2371) to R.A.F. The authors want to thank the technical support of Ms Chang Li He.
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3 To whom correspondence should be addressed. E-mail: rfissore{at}vasci.umass.edu
* Both these authors contributed equally to this work.
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Submitted on April 23, 2001; resubmitted on November 19, 2001; accepted on February 2, 2002.
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