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Strontium-induced rat egg activation

¹ Departments of Cell and Developmental Biology and ² Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat-Aviv 9978 Tel-Aviv, Israel

Correspondence should be addressed to R Shalgi; Email: shalgir{at}post.tau.ac.il

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca²⁺]_i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca²⁺]_i transient. Strontium chloride (SrCl₂) binds to and activates the Ca²⁺-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca²⁺]_i oscillations. Although SrCl₂ has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca²⁺]_i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl₂ (2, 4 or 6 mM) on [Ca²⁺]_i, by monitoring [Ca²⁺]_i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl₂ was optimal for inducing the first [Ca²⁺]_i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca²⁺]_i oscillations were lower and longer in SrCl₂-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl₂, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca²⁺]_i oscillations (8, 15 or 60 min in SrCl₂ respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl₂ caused a delay in CM. Our results demonstrate that SrCl₂ is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca²⁺]_i oscillations, CGE and CM.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The initial increase in cytosolic calcium concentration ([Ca²⁺]_i) occurring immediately after sperm–egg interaction at fertilization is necessary for triggering two majors events: cortical granule exocytosis (CGE) and stimulation of the cell cycle resumption to complete meiosis (CM), collectively known as egg activation (Whitaker & Larman 2001). This concept is supported by evidence derived from experiments performed with eggs of several species, including the rat (Raz & Shalgi 1998): (1) the actual increase in [Ca²⁺]_i within the fertilized egg cytoplasm, (2) the initiation of egg development by parthenogenetically activating a rise in [Ca²⁺]_i, and (3) the prevention of egg development initiation by suppression of the [Ca²⁺]_i rise (Kline & Kline 1992). The signaling pathway responsible for initiating and mediating [Ca²⁺]_i increase has been studied by several research groups. It has been shown that mammalian eggs express phospholipase C (Dupont et al. 1996, Mehlmann et al. 1998), which, when activated, hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messenger inositol 1,4,5- trisphosphate (InsP₃). InsP₃ interacts with InsP₃ receptors (InsP₃R) at the endoplasmic reticulum, thus inducing Ca²⁺ release (Patel et al. 1999). CM is mediated by a decrease in activity of both the cytostatic factor and the M-phase promoting factor, a heterodimer comprised of catalytic, CDK1, and regulatory, cyclin B1, components (Nixon et al. 2000, Carroll 2001). Recent studies have demonstrated that Ca²⁺ mediates the degradation of cyclin B1 by increasing the activity of an E3 ubiquitin ligase (Hyslop et al. 2004, Jones 2004).

Sperm–egg fusion initiates [Ca²⁺]_i oscillations within the egg that last several hours (Fissore et al. 1992). A fertilization-like [Ca²⁺]_i oscillation frequency dramatically increases the extent of parthenogenetic development of newly ovulated eggs, as compared with a single Ca²⁺ stimulus (Ozil & Huneau 2001), whereas an abnormally high frequency of oscillations results in cell death (Gordo et al. 2000). Some activators, such as ionomycin or ethanol, cause a single [Ca²⁺]_i transient, while others such as InsP₃, adenophostin A, thimerosal and strontium (SrCl₂) evoke repetitive [Ca²⁺]_i oscillations (Jellerette et al. 2000). SrCl₂ binds to and activates the Ca²⁺-binding site on the InsP₃R (Kline & Kline 1992, Marshall & Taylor 1994); more-over, SrCl₂-mediated [Ca²⁺]_i oscillations are generated via the InsP₃R (Brind et al. 2000). There are three known isoforms of InsP₃R that are expressed, to different extents, in various cell types. In mouse eggs, type I InsP₃R is by far the most abundant isoform; however, mRNAs of all isoforms are present within the egg (Parrington et al. 1998). The InsP₃Rs form tetramers at the membrane of the endoplasmic reticulum. The tetramers consist of a large cytoplasmic domain that contains the InsP₃ binding site, a membrane-spanning region that contains the Ca²⁺ channel, a small cytoplasmic C-terminus, and a large N-terminal cytoplasmic region that provides the main region for cytoplasmic regulators to act (Brind et al. 2000). InsP₃Rs are regulated by small modulators such as InsP₃, Ca²⁺ and ATP, as well as by protein–protein type interactions with large proteins like calmodulin and FKBP12 (Taylor 1998). Another mechanism for the long-term regulation of InsP₃R activity is its downregulation by proteolysis, probably by the proteasome (Brind et al. 2000).

SrCl₂ is a known activator of mouse eggs (O’Neill et al. 1991, Wakayama et al. 1998, Kishikawa et al. 1999, Otaegui et al. 1999), but little is known regarding its effectiveness in activating rat eggs. It has recently been reported that SrCl₂ effectively activated rat eggs and triggered development to the blastocyst stage (Krivokharchenko et al. 2003). However, no information was presented regarding the dynamics of [Ca²⁺]_i and its effect on the early events of egg activation (CGE, CM).

SrCl₂ provides a simple yet tightly-controlled technique of egg activation. It is added directly to the medium, without the need for microinjection and the possible damage to the egg membrane, whereas the extent of [Ca²⁺]_i signal and its duration are easily controlled by SrCl₂ concentration and time of exposure. In the current study, we monitored [Ca²⁺]_i changes during incubation with various SrCl₂ concentrations and established the optimal concentration for the occurrence of CGE and of CM. We also studied the effect of the number of SrCl₂-induced [Ca²⁺]_i transients on CGE and CM. Although SrCl₂ is extensively used as a parthenogenetic activator of eggs in a number of species (Tateno & Kamiguchi 1997, Okada et al. 2003) and its action has been described in considerable detail in the mouse egg (Kline & Kline 1992, Bos-Mikich et al. 1993), the present report is the first comprehensive study relating SrCl₂ parthenogenetic activation of rat eggs to the early events of fertilization - [Ca²⁺]_i dynamics, CGE and CM.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Animals
Wistar-derived rats were housed in air-conditioned, light-controlled rooms, with food and water available ad libitum. Twenty-three- to twenty-five-day-old female rats were primed with a subcutaneous injection of 10 IU pregnant mare’s serum gonadotropin (PMSG; Syncro-Part, Sanofi, Paris, France). An intraperitoneal injection of 10 IU human chorionic gonadotropin (hCG; Sigma Chemical Co., St Louis, MO, USA) was administered 48–54 h after PMSG.

Collection of eggs
Ovulated eggs at metaphase II (MII) were isolated, 14 h after hCG injection, from the oviductal ampullae into regular or Ca²⁺- and Mg²⁺-free culture medium (Toyoda HEPES (TH or TH – /–) supplemented with 0.4% BSA; Talmor et al. 1998). Cumulus cells were removed by hyaluronidase (400 IU/ml, Sigma). For in vitro fertilization, eggs were treated with -chemotrypsin (50 µg/ml, Sigma) to remove the zona pellucida (ZP) prior to insemination. All manipulations were performed on a warm plate (37 °C).

Parthenogenetic activation
Eggs were parthenogenetically activated by either calcium ionophore (ionomycin 407950; Calbiochem, San Diego, CA, USA) or SrCl₂. Aliquots of 4 mM ionomycin in dimethylsulfoxide (DMSO) were kept at –70 °C and diluted to the final concentration of 2 µM just before use in TH – /– medium. Freshly prepared SrCl₂ (2, 4 or 6 mM in TH – /– medium) was used. MII eggs were incubated for 5 min in the presence of ionomycin and then for 0, 10, 25, 40, 55 or 70 min in fresh TH medium without ionomycin. Other batches of MII eggs were incubated for 8, 15 or 60 min in the presence of various concentrations of SrCl₂ followed by additional incubation in SrCl₂-free TH medium, up to a total incubation period of 15, 30, 45, 60 or 75 min. For details, see the Results section.

In vivo/in vitro fertilization
PMSG- and hCG-primed female rats were allowed to mate overnight with males of proven fertility. In vivo fertilized eggs were isolated from the oviductal ampullae of animals, 15.5 h after hCG administration, into TH medium, and their cumulus cells were removed as described earlier for MII eggs.

For in vitro fertilization, spermatozoa were collected from the uteri shortly after mating, and diluted in rat fertilization medium (Ben-Yosef et al. 1993) to a final concentration of 0.7 to 1.3 x 10⁶ spermatozoa/ml. Insemination of ZP-free eggs by capacitated sperm was performed in a thermostatic chamber, suitable for [Ca²⁺]_i measurements, as previously described (Ben-Yosef et al. 1996).

Measurement of [Ca²⁺]_i
To follow [Ca²⁺]_i changes, MII eggs, either ZP-enclosed or ZP-free, were collected as described and loaded with the Ca²⁺-sensitive dye, fura-2-AM (3 µM; Molecular Probes, Eugene, OR, USA), for 30 min in TH medium at 37 °C. Eggs were washed free of the dye and allowed to attach to a poly-L-lysine coated coverslip, in TH – /– medium, under mineral oil. The coverslip was placed in a thermostatic chamber adjusted to 37 ± 1 °C. Free [Ca²⁺]_i was determined by monitoring the fluorescence ratio at 340/380 nm using an inverted microscope (Nikon TMD; Nikon Corp, Tokyo, Japan), attached to an imaging workstation, controlled by Metamorph and Metafluor software (Universal Imaging, Downingston, PA, USA).

DNA staining and CGE quantification
Eggs were fixed in 3% paraformaldehyde, stained with Lens culinaris agglutinin (LCA; Vector, Burlingame, CA, USA) and costained with Texas-red streptavidin (Vector) for detection of CGE (Eliyahu & Shalgi 2002) and labeled with Hoechst 33342 (Sigma) for determining cell cycle and fertilization status (Shalgi & Phillips 1988). Labeled eggs were visualized and photographed with a Zeiss confocal laser-scanning microscope (LSM). The Zeiss LSM 410 (Oberkochen, Germany) is equipped with a UV laser (Coherent Inc. Santa Clara, CA, USA) and with a 25 mW krypton-argon laser and a 10 mW helium-neon laser (488, 543, and 633 maximum lines). A 40 x NA/1.2 planapo-chromat water-immersion lens (Axiovert 135 M, Zeiss) was used for all imaging. Confocal micrographs of 3–4 eggs from each experimental group were analyzed by densitometry. The staining intensity was calculated using the corrected mean density values obtained by the LSM software (Abbott et al. 1999).

Statistical analysis
Data were evaluated by one-way ANOVA, and differences between treatment groups were determined by using a Chi-square test; P < 0.01 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The effect of SrCl₂ concentration on [Ca²⁺]_i oscillations
A very early observable event occurring after sperm–egg interaction or after parthenogenetic activation is an increase in [Ca²⁺]_i, which leads to the early events of egg activation (i.e. CGE and CM). We examined the amplitude and the duration of [Ca²⁺]_i oscillations in MII triggered by 2, 4 or 6 mM SrCl₂ and compared them to those caused by fertilization or by ionomycin (Table 1). All three concentrations of SrCl₂ triggered an initial [Ca²⁺]_i transient that was followed by a series of [Ca²⁺]_i oscillations of shorter duration and lower amplitude. The oscillations in individual eggs were repetitive with regular peak-to-peak intervals. Eggs treated with either 2 or 4 mM SrCl₂ exhibited no significant differences in the duration or frequency of Ca²⁺ oscillations, which were similar to those observed during in vitro fertilization. The amplitude of the initial [Ca²⁺]_i transient induced by SrCl₂ (regardless of concentration) was higher than that of the following [Ca²⁺]_i oscillations (Fig. 1B–D), whereas the amplitude of the first sperm-induced transient was lower than the following [Ca²⁺]_i oscillations (P < 0.01; Fig. 1A). The oscillations induced by SrCl₂ were of longer duration (i.e. they had longer peak-to-peak intervals) than those induced by sperm (Fig. 1A–D). The duration and amplitude of the first [Ca²⁺]_i transient induced by 6 mM SrCl₂ were higher and significantly longer than those induced by either 2 or 4 mM SrCl₂ or those exhibited during fertilization (P < 0.01; Table 1, Fig. 1B–D). As expected, repetitive oscillations continued as long as SrCl₂ was present in the culture medium (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1 Fertilization and SrCl2-induced Ca2+ oscillations. [Ca2+]i in fura-2AM-loaded rat eggs that were either fertilized in vitro (A) or parthenogenetically activated by 2 mM (B), 4 mM (C) or 6 mM (D) SrCl2.

The effect on egg activation of a single [Ca²⁺]_i transient, induced by either SrCl₂ or ionomycin
We evaluated the effect on CM of a single [Ca²⁺]_i transient induced by SrCl₂. As presented in Table 1, the second [Ca²⁺]_i transient, in SrCl₂-activated eggs, occurs 12.2 ± 3.2 min post exposure to SrCl₂. We thus exposed the eggs for 8 min to 2, 4 or 6 mM SrCl₂ in TH – /– medium followed by incubation in TH medium. After 30 min in culture (8 min in SrCl₂ and 22 additional min in TH), more than half of the eggs reached anaphase in a dose-dependent manner (53%, 78% or 88% induced by 2, 4 or 6 mM SrCl₂ respectively; P < 0.01), whereas 45 min were sufficient for 31–39% of the eggs to reach telophase at all SrCl₂ concentrations tested. After 60 min, 56% of the eggs induced by 4 mM SrCl₂ extruded polar body (PBII) while only 42% and 34% of the eggs were induced to do so by 6 or by 2 mM SrCl₂ respectively (P < 0.01; Fig. 2). The membrane of most of the eggs that were activated by 6 mM SrCl₂ was undulated (light microscopy; data not shown). CM induced by 1 mM SrCl₂ was very slow - only a small percentage of eggs extruded PBII after 75 min in culture (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 2 Completion of meiosis induced by 2, 4 or 6 mM SrCl2. Eggs were collected 14 h after hCG administration and activated by exposure to 2, 4 or 6 mM SrCl2. MII eggs were exposed for 8 min to 2 mM (A), 4 mM (B) or 6 mM (C) SrCl2 in TH – /– medium, washed and cultured in TH medium for additional time periods to complete a total of 15, 30, 45 or 60 min in culture. Each graph represents 9 experiments, performed on different days. At least 25 eggs were examined at each time point on each experimental day. Time (min) indicates total time in culture. Eggs (%) indicates the percent of eggs at MII (), anaphase (), telophase () and PBII (X).

View larger version (19K): [in this window] [in a new window]   Figure 3 Completion of meiosis induced by either ionomycin or SrCl2. Eggs were collected 14 h after hCG administration and activated by exposure to 2 mM SrCl2 for 8 min (A) or to 2 µM ionomycin for 5 min (B) in TH – /– medium, washed and cultured for additional time periods in TH medium to complete 15, 30, 45, 60 or 75 min in culture. Each graph represents 5 experiments performed on different days. At least 25 eggs were examined at each time point on each experimental day. Time (minutes) indicates total time in culture. Eggs (%) indicates the percent of eggs at MII (), anaphase (), telophase () and PBII (X).

View larger version (67K): [in this window] [in a new window]   Figure 4 CGE in eggs induced by SrCl2, ionomycin or sperm. A–D, light microscope; A'–D', fluorescence microscope. Representative confocal micrographs of eggs, colabeled for CGE (red) and DNA (blue) are shown. MII; metaphase II eggs were cultured for 8 min in TH – /– medium devoid of activators followed by 37 min in TH medium (control; A, A’). SrCl2: eggs were cultured for 8 min in 2 mM SrCl2 in TH – /– medium followed by 37 min in TH medium (B, B’). Ionomycin: eggs were cultured for 5 min in 2 µM ionomycin in TH – /– medium followed by 40 min in TH medium (C, C’). Fertilization: in vivo fertilized egg (D, D’), arrow indicates the sperm head. Quantification of CGE intensity is presented in Table 2. Scale bar = 10 µm.

SrCl₂ (2 mM) caused [Ca²⁺]_i oscillations with a 6.6 min peak-to-peak interval (Table 1). We exposed the eggs to 2 mM SrCl₂ for 8, 15 or 60 min, which was expected to induce 1, 2 or 10 oscillations respectively, and then transferred them into TH medium to complete a period of 60 min in culture. The eggs were fixed and labeled for CGE and for DNA. Longer exposure to SrCl₂ (15 vs 8 min) correlated with a higher rate of PBII extrusion (44% vs 34% respectively; P < 0.01). However, eggs exposed to SrCl₂ for 60 min proceeded from MII to anaphase (34%) and telophase (36%), but only 18% extruded PBII (P < 0.01; Fig. 5).

View larger version (18K): [in this window] [in a new window]   Figure 5 The effect on the progression of CM incubation in SrCl2. Eggs were cultured in the presence of 2 mM SrCl2 in TH – /– medium for 8, 15 or 60 min and were then transferred to TH medium to complete a period of 60 min in culture. Each column represents at least 3 experiments performed on different days. At least 25 eggs were examined at each time point at each experimental day (P < 0.01). Time (minutes) indicates the duration of exposure to SrCl2. Eggs (%) indicates the percent of eggs at MII (white section), anaphase (black section), telophase (dark gray section), and PBII (light gray section) after 60 min culture period.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
SrCl₂ is a commonly employed agent for induction of parthenogenetic activation of mouse eggs. Some researchers reported that SrCl₂ triggers a series of [Ca²⁺]_i oscillations in mature MII mouse (Kline & Kline 1992, Bos-Mikich et al. 1993), pig (Okada et al. 2003) and hamster (Tateno & Kamiguchi 1997) eggs and is able to cause egg activation. In a recent study, the efficiency of 2 mM SrCl₂ for parthenogenetic development of rat eggs was tested, but no details were reported regarding its ability to cause [Ca²⁺]_i oscillations, CGE or PBII extrusion (Krivokharchenko et al. 2003). The present study examines the effect of SrCl₂ on the early temporal correlation among these three parameters of egg activation.

SrCl₂, as well as many other parthenogenetic activators (InsP₃, sperm factor, thimerosal, adenophostin A) induces repetitive and regular [Ca²⁺]_i oscillations and mimics sperm-induced calcium dynamics. Despite the superficial similarity of the overall phenomena, there are marked differences in the pattern of [Ca²⁺]_i changes among the different parthenogenetic stimuli (e.g. Jellerette et al. 2000). Thus, for example, in the rat egg SrCl₂ induces a greater first calcium transient, followed by oscillations that are lower in amplitude, more prolonged and of lower frequency than those induced by sperm. By comparison, ionomycin induces only a single calcium transient. Nevertheless, all these agents can cause egg activation up to PBII extrusion. Krivokharchenko et al.(2003) recently reported that ethanol or SrCl₂ treatments produced similar success rates of rat embryo implantation. In view of these reports and our own data presented here, what is the importance of the orderly pattern of calcium oscillations induced by sperm and mimicked to a varying degree by other parthenogenetic agents? Are they necessary for the efficient block of polyspermy? Do they support better success rates of activation progression and, ultimately, better rates of implantation?

The only definitive experimental evidence, correlating electropermeabilization-induced calcium oscillations in mouse eggs with 50% CGE (4 transients) or pronuclei formation (8–24 transients), was reported by Ducibella et al.(2002). Varying the duration of SrCl₂ exposure, we could control the number of transients (1, 2 or 10), yet found there was little difference in the intensity of CGE or the progression to PBII extrusion; these parameters were comparable to those induced by sperm. Actually, 1 or 2 transients exhibited a better rate of PBII extrusion than 10 oscillations, possibly reflecting the toxic effects of prolonged exposure to SrCl₂. The differences between the results of Ducibella et al.(2002) and those reported here may be attributed to differences between methods causing a different pattern of [Ca²⁺]_i oscillations or differences in species studied.

Although both 2 µM ionomycin and 8-min exposure to 2 mM SrCl₂ cause a single [Ca²⁺]_i transient, the ionomycin-induced transient is of much lower amplitude. Interestingly, ionomycin evokes a much lower CGE response (see Table 2), but a similar degree of PBII extrusion (Fig. 2). This could be interpreted in terms of two independent processes being initiated by the first [Ca²⁺]_i transient: CGE and CM. The hypothesis that an increase in [Ca²⁺]_i is required for CGE is well established (Kline & Kline et al. 1992, Raz et al. 1998). It is possible that the high transient resulting from SrCl₂ exposure, but not from ionomycin, activates Ca²⁺-dependent proteins such as Ca²⁺-dependent protein kinase C, which, in turn, affect the degree of CGE (Bos-Mikich et al. 1995, Jellerette et al. 2000). It should be noted that Raz et al.(1998), using ionomycin and BAPTA-AM (to buffer cytosolic [Ca²⁺]_i), reported that CGE was more sensitive than PBII extrusion to [Ca²⁺]_i concentrations. Their experiments, however, were performed in Ca²⁺-containing medium and the influx of extracellular calcium through the ionophore could account for these differences.

In conclusion, the current study demonstrated that SrCl₂ can be used as an easily controlled parthenogenetic agent, which mimics fairly faithfully the early events of egg activation ([Ca²⁺]_i increase, CM and CGE). Our results, as well as those reported by others, suggest that a single large calcium transient is sufficient for the orderly progression of the egg through these early events. The role of the subsequent transients in the sperm-activated egg is less clear and merits further study.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We gratefully thank Dr Leonid Mittelman for his excellent technical assistance at the confocal microscope.

This work is in partial fulfillment of the requirements for the PhD degree of R Tomashov-Matar at the Sackler Faculty of Medicine, Tel-Aviv University. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 29 March 2005
First decision 23 May 2005
Revised manuscript received 2 June 2005
Accepted 21 June 2005

R Tomashov-Matar and D Tchetchik contributed equally to this work

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