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The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome

Monash Immunology and Stem Cell Laboratories, Level 3, STRIP 1- Building 75, Monash University, Wellington Rd., Clayton, Australia, 3800

Correspondence should be addressed to O Lacham-Kaplan; Email: Orly.Lacham-kaplan{at}med.monash.edu.au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The present research investigated the effects of various strontium concentrations, in combination with different incubation periods, on mouse parthenogentic oocyte activation and blastocyst development. The results for blastocyst development showed a trend indicating that 10 mM strontium for 3 h was the optimal strontium protocol. Ethanol, an agent that incites oocyte activation via a monotonic rise in calcium, was employed as a control. The outcome of blastocyst formation arising from parthenogenic ethanol activation was significantly less (P < 0.001) than that achieved by the optimal strontium protocol. To assess the impact of strontium oocyte activation on embryo viability following fertilization with immature germ cells, the protocol of 10 mM strontium for 3 h was applied to oocytes injected with round spermatids and then compared with other protocols. The results indicate that following round-spermatid injection the benefits derived from strontium artificial oocyte activation are evident during both pre- and post-implantation development. However, in order to adjust the protocol to the most effective round-spermatid injection in relation to the oocyte cell cycle, injection was done 1.5 h after strontium activation followed by another 1.5 h activation in strontium. The implementation of round-spermatid injection in combination with this oocyte-activation protocol led to live-birth outcomes not significantly different to those outcomes obtained by mature spermatozoa.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Oocyte activation is not a sole event but a series of occurrences that initiate the progression of the oocyte from a haploid fertilized cell to a zygote. Mammalian oocyte activation, in nature triggered by fertilization, allows the oocyte to resume meiosis and proceed to embryonic development (Carroll 2001). Meiosis is completed with the extrusion of the second polar body and the resulting haploid chromosomal state of the oocyte.

Artificially recreating the events of oocyte activation has become an important component of assisted reproduction technology. To allow for the release of the oocyte from metaphase II (MII) arrest, effective artificial oocyte activation is critical unto itself but it is also critical because of the impact it has on later developmental events (Ozil & Huneau 2001). Suboptimal artificial oocyte activation is thought to contribute to disappointing outcomes experienced by many procedures for assisted reproduction technology (Alberio et al. 2001).

The earliest notable event in oocyte activation, in all studied species, is an increase in the level of intracellular calcium (Swann & Ozil 1994). In mammals, the levels of intracellular calcium oscillates continuously throughout oocyte activation (Cuthbertson & Cobbold 1985, Miyazaki et al. 1992), continuing until pronucleus formation (Jones et al. 1995). It has been suggested that calcium oscillations coordinate the many events that occur throughout fertilization (Ben-Yosef & Shalgi 2001) and that each calcium oscillation incrementally propels the events of oocyte activation (Ducibella et al. 2002). Oocyte activation can, however, occur with a single rise in calcium, so the full role of calcium oscillations is unclear (Swann & Ozil 1994). An association has been identified between calcium oscillations and superior blastocyst composition (Bos-Mikich et al. 1997). Additionally, the pattern, frequency, and amplitude of early calcium oscillations impact on post-implantation development (Ozil & Huneau 2001). Prevention of calcium oscillations completing their full course during fertilization will retard pronucleus formation (Lawrence et al. 1998) and calcium oscillations of abnormal frequencies will induce development arrest (Gordo et al. 2002).

The paucity of knowledge surrounding the events of oocyte activation present some challenges for artificial oocyte activation. As calcium is recognized as the most known fundamental factor of oocyte activation, many approaches to artificially induce oocyte activation have focused on recreating increases in intracellular calcium. Methods to induce this include the direct injection of calcium into the oocyte (Machaty et al. 1996), an electrical pulse that through phospholipid destabilization creates pores for the influx of extracellular calcium (Sasagawa & Yanagimachi 1996), isolation and injection of the sperm factor thought to be responsible for oocyte activation at fertilization (Fissore et al. 1998), employment of adenophostin A, an inositol trisphosphate (InsP₃) receptor agonist, (Brind et al. 2000), promoting of extracelluar calcium entry via the use of ethanol (Cuthbertson & Cobbold 1985), and inducing calcium oscillations by employing the divalent cation, strontium (Kline & Kline 1992). Alternatively, protein inhibitors such as cycloheximide bypass an intracellular rise in calcium and directly induce meiosis by undermining cyclin B and therefore maturation promoting factor (MPF) (Bos-Mikich et al. 1995). While these may be credible methods for re-enacting oocyte activation, none are able to achieve this with the efficiency provided by spermatozoa.

In the mouse, strontium has been successfully employed in many studies to induce artificial oocyte activation. The ability of strontium to provoke calcium oscillations appears to be more physiologically sound than alternative methods of oocyte activation that produce a monotonic rise in calcium. Calcium oscillations induced by strontium lead to improved blastocyst composition (Bos-Mikich et al. 1997) and superior pre- implantation development (Lacham-Kaplan et al. 2003). The aptitude of strontium has lent itself to satisfactory outcomes in a range of mouse reproductive technologies; however, there is a paucity of research that has investigated how strontium is best applied. Therefore, the potential to exploit the benefits of strontium may be thwarted by a lack of insight concerning its optimal employment.

The aim of the present study was to compare various strontium protocols with the intention of revealing an optimal strontium strength and incubation time combination. The research also intended to compare strontium activation with ethanol, an agent that activates oocytes through the production of a monotonic rise in calcium. The development of artificially activated oocytes was compared with that of in vitro-fertilized oocytes. In addition, we examined the effects of these activation protocols on pre- and post-implantation embryo development following injection of immature male germ cells into MII oocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Ethics
All experiments were approved by the Monash University Animal Ethics Committee under application number 2003/05. Experiments were conducted in accordance with the Australian National Health and Medical Research Council (NIH & MRC) code of practice for the care and use of animals for scientific purposes.

Oocyte collection
Hybrid F₁ female mice (C57BL female x CBA male) at 4–6 weeks of age were superovulated by subcutaneous injection of 10 IU pregnant mares’ serum gonadotrophin (Intervet, Sydney, Australia) followed by subcutaneous injection of 10 IU human chorionic gonadotrophin (Intervet) 48–50 h later. Female mice were killed by cervical dislocation 13–14 h after injection of human chorionic gonadotrophin. The oocytes were released from the oviducts into M2 handling medium containing 100 IU/ml hyaluronidase (type IV-S; Sigma Chemical Co., St Louis, MO, USA) for no longer than 5 min to remove cumulus cells. Morphologically normal MII oocytes were washed in and transferred to warm and equilibrated M16 culture medium and placed into incubation under 5% CO₂ in air at 37 °C for 20 min before they were used for experiments.

Oocyte activation with strontium
The amounts of SrCl₂ (Sigma) that were required for different-strength solutions were dissolved in sterile deionized water (JDH Biosciences, Lenexa, KS, USA) to give 10 x stock solution. For each experiment, fresh strontium culture medium was prepared. A volume of 0.1 µl prepared stock solution was placed into an Eppendorf tube (Greiner Labortechnik, Frikenhaussen, Germany) containing 0.99 µl calcium-free M16. Microdroplets (20 µl) were arranged onto a 35 mm Petri dish (Falcon, Franklin Lakes, NJ, USA), covered with mineral oil (Sigma) and incubated under 5% CO₂ in air at 37 °C for 30 min. For activation, oocytes were washed twice, cultured in equilibrated strontium solution, and left for the allotted amount of time. Following the oocyte activation period, oocytes were washed twice in Ca²⁺/M16 where they remained for culture under mineral oil in an atmosphere of 5% CO₂ in air at 37 °C.

Oocyte activation with ethanol
A 50 µl droplet of 8% ethanol (BDH, Poole, Dorset, England) in M2 handling medium was placed onto a 35 mm Petri dish (Falcon) and covered with mineral oil. Oocytes were transferred into 8% ethanol for 5 min exactly Once removed oocytes were washed three times in pre-equilibrated M16 in which they remained for culture under mineral oil in an atmosphere of 5% CO₂ in air at 37 °C.

Sperm and round-spermatid collection
Hybrid F₁ male mice (C57BL female x CBA male) at 8–12 weeks of age were killed by cervical dislocation. The testes and cauda epididymis were dissected out from the rest of the reproductive tract. An incision was made in the testis capsule to allow for the removal of the seminiferous tubules into 2 ml previously equilibrated modified T6 (MT6) medium. The content of the seminiferous tubules were liberated by mechanical agitation with forceps, leaving a suspension of cells including round spermatids, which were used immediately for the experiments.

For spermatozoa, a small incision was made in the cauda before placing it in 2 ml of equilibrated MT6 medium. The tissue was removed 1 h later. Spermatozoa were incubated under 5% CO₂ in air at 37 °C and left undisturbed for 2 h for capacitation before they were used for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

IVF
Approximately 20 oocytes were introduced into a sperm solution of a concentration of (3–5) x 10⁶ sperm/ml. After 2.5 h of co-incubation with sperm, the oocytes were removed from the MT6 medium, washed three times and cultured in equilibrated M16 medium at 37 °C in an atmosphere of 5% CO₂ in air. Oocytes were assessed approximately 6 h following IVF treatment for the presence of a second polar body and two pronuclei. The presence of such confirmed fertilization. Zygotes were isolated and their development assessed every 24 h up to 5 days.

ICSI and round-spermatid injection (ROSI)
A microinjection procedure followed that described in detail that by Lacham-Kaplan et al.(2003). Oocytes were injected using a fine glass capillary with a 5 µm internal diameter, attached to a Piezo microinjection system (PiezoDrill; Burleigh Instruments, Burleigh Park, Fishers, NY, USA). Before injection, the heads of the sperm were separated from the tails by applying a strong Piezo pulse on the neck region. Sperm heads were aspirated and injected individually into the oocytes. Round spermatids were identified by size and morphology as described by Kimura & Yanagimachi (1995). The selected cells were individually drawn into an injection pipette and through repeated aspiration the nucleus was isolated from the rest of the cell before injected into the oocytes. Following microinjection, oocytes which survived the injection were into M16 for incubation under 5% CO₂ in air at 37 °C. Confirmation of fertilization and embryo development of oocytes following ICSI or ROSI was undertaken as described above for IVF.

Statistical analysis
The data of activation, fertilization and embryo development to the blastocyst stage were analysed by ² test using Yates’ correction and analysis of variance tests as appropriate. The total number of fertilized and activated oocytes and blastocysts were compared between different treatment groups. If the P value was less than or equal to 0.05 the difference was regarded as significant. ² tests were also used to compare the total number of pups resulting from embryo transfer.

Experimental design
For all experiments there was a minimum of five repeats, with each group being assigned a total of 100 oocytes. Following treatment, oocytes underwent two or three washes in Ca²⁺/M16 20 µl droplets under mineral oil, before incubation under 5% CO₂ in air at 37 °C. Once activation and fertilization were established the numbers of two-cell, four-cell, morulae and blastocyst embryos were recorded up until day 5. Development was assessed every 24 h at a time dictated by the time of oocyte activation or fertilization on day 1.

The experiments in this study were as follows. Initially, oocytes were exposed to strontium – 1.74, 5, 10 or 15 mM – and cultured for 1.5, 3, 6 or 12 h; resulting in 16 different strontium protocols for comparison. The comparisons were done between different concentrations for each time point. Within each time frame, three control groups were implemented: an ethanol-treatment group, an IVF group and a vehicle control of Ca²⁺/M16 containing no strontium. Following activation treated oocytes were incubated in Ca²⁺/M16 for further development. Oocyte activation and development through to the blastocyst stage were assessed every 24 h from activation and fertilization.

Based on the outcome of the first experiments, oocytes were exposed to a series of strontium concentrations. The oocytes were exposed to a high concentration of strontium, followed by culture in a lower concentration of strontium. The following combinations that were employed were (a) 10 mM strontium for 1.5 h followed by 1.74 mM strontium for 1.5 h, (b) 10 mM strontium for 3 h followed by 1.74 mM strontium for 3 h and (c) 10 mM strontium for 3 h followed by 1.74 mM strontium for 6 h. The best outcome from the first experiments was used as a control for these protocols. Following activation treatments oocytes were incubated in Ca²⁺/M16 for a further development. Oocyte activation and development through to the blastocyst stage were assessed every 24 h from activation and fertilization.

Effective activation protocols identified were used in combination with ROSI. It was anticipated that the implementation of ROSI following second polar-body extrusion might necessitate an interruption to the employment of strontium culture. The effect of this interruption on blastocyst development was explored. Firstly, the timing of a second polar-body extrusion following artificial oocyte activation was investigated. Oocytes were activated by the optimal strontium protocol, or by ethanol, and examined for the presence of a second polar body at 30, 60, 90 and 120 min from the commencement of activation. Following second polar-body extrusion, oocytes were removed from the strontium culture medium and placed into Ca²⁺/M2 for 30 min, mimicking the break required for manipulation. Oocytes were then placed back into strontium culture for the time required to give a total time in the best concentration of strontium as identified in experiments 1 and 2. As a control, a group of oocytes was activated using the most optimal protocol identified in experiments 1 and 2 with no interruption. Following treatment, oocytes were washed in Ca²⁺/M16 and incubated under 5% CO₂ in air at 37 °C. Development was assessed until day 5. Once the impact of a break in strontium culture was established, oocytes prior to, or following, ROSI were subjected to different activation protocols. Each protocol amounted to an overall time of the best protocol and the optimal concentration identified. These protocols were as follows: (a) ROSI in Ca²⁺/M2 followed by incubation in 10 mM Sr²⁺/M16 for 3 h; (b) exposure to 10 mM Sr²⁺/M16 for 1.5 h followed by ROSI in Ca²⁺/M2 followed by incubation in 10 mM Sr²⁺/M16 for 1.5 h; (c) ROSI in Ca²⁺/M2 followed by activation in 8% ethanol for 5 min; (d) exposure to 8% ethanol for 5 min followed by ROSI in Ca²⁺/M2 1 h later; (e) ICSI (control). Following treatment, oocytes were washed and cultured in Ca²⁺/M16. Fertilization was assessed 6 h later and development was assessed until day 5.

To identify the effects strontium activation has on in vivo embryo development, embryos at the two-cell stage were transferred to pseudopregnant female on day 1 of pregnancy. The most favourable strontium and ethanol protocols from experiment 3 as indicated by blastocyst outcomes were implemented to activate oocytes to produce two-cell ROSI embryos for transfer. Pregnant females were allowed to give birth. The number of pups born was recorded and their development up to 4 weeks after birth was monitored. A random sample of pups, two of each sex, from each of the strontium ROSI, ethanol ROSI and ICSI groups, were used for fertility assessment. At 6 weeks of age, F₂ female offspring were allowed to mate with 8-week-old F₁ male mice. At 8 weeks of age F₂ male offspring were allowed to mate with 6-week-old F₁ females. The number of foetuses was examined on day 15 of pregnancy.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
For all experiments, there was a minimum of five repeats, with each group being assigned a total of 100 oocytes.

Percentages of blastocysts are indicated from the number of oocytes activated or fertilized (Table 1, Fig. 1). Each of the strontium and ethanol treatment groups produced high rates of oocyte activation and no statistical difference between treatments were found. Development to blastocysts in the control group following IVF was significantly higher (P < 0.001) than all oocyte-activation treatments. Progression to the blastocyst stage was not significantly different between strontium treatment groups within any of the time groups. Overall, incubation in 10 mM strontium for 3 h produced the highest blastocyst rate of 39%. This group was therefore used as a control group for following experiments.

View larger version (43K): [in this window] [in a new window]   Figure 1 Overall embryo developmental outcomes following oocyte activation in different strontium concentrations (mM) for different time periods. (A) 1.5 h, (B) 3 h, (C) 6 h, (D) 12 h C, cells.

In all activation groups investigated, the decline in development was evident between the four-cell to the eight-cell/morula stage (Fig. 1). Whereas activation with strontium produced high activation followed with a gradual reduction in development similar in all strontium-treatment groups, ethanol activation found less reliable. Development to the two- and four-cell stages was not consistent even though the method used was similar for all experiments. Nonetheless, development to blastocysts was lower than that obtained by strontium at any concentration regardless of whether the development to two- and four-cell stages was high.

Exposing the oocytes to high strontium concentrations followed by lower ones did not have any positive effect on blastocyst development (Table 2). Hence treatment of oocytes for 3 h in 10 mM strontium was still superior to any combination of two different strontium concentrations.

The results from ROSI using strontium activation and ethanol activation before and after extrusion of the polar body are summarized in Table 3. Development to the blastocyst stage was higher when ROSI was performed after the extrusion of the polar body. This was 1 h after ethanol activation and 1.5 h after strontium activation. The best blastocyst development of 58% was achieved when ROSI was performed after the oocytes extruded their polar body. In this protocol oocytes were activated for 1.5 h followed by ROSI and a continuation of activation in 10 mM strontium for a further 1.5 h. This group was also the only group, which was not significantly different to the ICSI group.

View larger version (22K): [in this window] [in a new window]   Figure 2 Births following embryo transfers of two-cell (2-c) embryos from ROSI embryos resulted from strontium activation and from ethanol (Ethol) activation, and from ICSI. Letters above the bars: a is significant to b (P < 0.001).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The purpose of this study was to identify the optimal strontium protocol to activate mouse oocytes. To explore this, different protocols that encompassed a range of strontium dosage and time-exposure combinations were used. Assessment of oocyte activation as the presence of a second polar body and a single pronucleus indicated that each of the groups explored demonstrated a similar ability to activate oocytes. It has been suggested, however, that proper indication of oocyte activation extends beyond second polar-body and pronucleus formation (Lacham-Kaplan et al. 2003). Therefore, development to the blastocyst stage was examined as a more complete indicator of oocyte activation capability and to help distinguish between treatments.

It appears that blastocyst development reached an optimal time exposure for each strontium concentration and declined once this optimal time exposure was exceeded (Fig. 1). For the lowest strontium concentrations explored (1.74 mM), 6 h was optimum while for moderate dosages (5 and 10 mM) of strontium a 3 h time period was optimum and for high concentrations (15 mM) a 1.5 h time period was optimal, with the best outcome resulting from exposure of oocytes to 10 mM strontium for 3 h. Nonetheless, the different combinations of time and concentration treatments revealed that exposure of mouse oocytes to strontium for 1.5 h is not an effective treatment to result in optimal blastocyst development and longer than 6 h found detrimental to embryo development. Exposure to calcium transients (Ozil et al. 2005) for durations of between 15 and 50 min was not different in inducing activation and development to blastocysts when electropermeabilization at 2-min intervals was used. Strontium promotes calcium oscillations through InsP₃ receptors (Zhang et al. 2005), which take longer time to activate, and hence require longer periods of time to be effective. This may also explain why polar-body extrusion after strontium treatment occurred later (1.5 h) than that observed following ethanol activation (1 h). Following the exposure to ethanol, an intracellular rise in calcium is attained by facilitating an increase in oocyte membrane permeability to calcium and thereby permitting entry of extracellular calcium into the cell (Cuthbertson & Cobbold 1985). Mobilization of intracellular calcium is also believed to contribute to the monotonic rise in calcium by ethanol activation (Shiina et al. 1993). Thus it is likely that both extracellular and intracellular calcium similar to electropermeabilization are drawn upon to reach the intracellular calcium threshold level required to activate quicker than strontium. Ethanol activation induces a high incidence of aneuploidy in haploid embryos following artificial oocyte activation with ethanol (O’Neill & Kaufman 1989) due to promoting premature postovulatory aging resulting in spindle organization errors and thereby chromosome malsegregation. Data from the present study also show that embryonic development to the two- and four-cell stages following ethanol activation is not consistent, contributing to the fact that ethanol is not beneficial for mouse oocyte activation.

The minimum dosage required to induce calcium oscillations in mouse oocytes is unknown. While Cheek et al.(1993) reported that 8 mM strontium is not enough to induce calcium oscillations in mouse oocytes, Kline & Kline (1992) identified 4.6 mM strontium as able to induce oocytes oscillations and 10 mM strontium for 2 h to induce calcium oscillations that were similar to those induced by spermatozoa but lower in frequency and amplitude. Bos-Mikich et al.(1995) also suggested that 10 mM strontium is the optimal dosage to induce calcium oscillations in mouse oocytes to result in optimal embryo development. In the present study calcium oscillations were not measured, and therefore it cannot be certain which strontium concentrations were able to stimulate calcium oscillations at sufficient levels to support optimal embryo development By using ROSI we have been able to explore whether the findings of 10 mM for 3 h exposure can be extrapolated into improved pre-implantation developmental outcomes in a diploid model and whether this will translate to have a positive impact on post-implantation development.

Round spermatids are the most immature germ cells to have acquired a haploid set of chromosomes (Lacham-Kaplan & Trounson 1997). Their DNA status is identical to mature spermatozoa, making them attractive replacements for spermatozoa. Aslam et al.(1998) has reported that of the 648 ROSI attempts worldwide only nine normal deliveries have occurred, indicating that while potentially viable, this is also a highly problematic technique. This has led to the advocacy of complete dismissal of ROSI (Silber et al. 1997) and to the procedure being completely banned in the UK (Ezeh et al. 1999). In the context of disappointing outcomes, critiques of ROSI’s failings have ensued (Devroey 1998, Silber & Johnson 1998, Vanderzwalmen et al. 1998), with focus being placed upon oocyte activation inadequacies in addition to genetic concerns.

Suboptimal oocyte activation may be responsible for the low success rates of clinical ROSI (Yanagimachi 2001). Human round spermatids are generally capable of inducing effective oocyte activation but the employment of round spermatids originating from men with spermiogenesis failure leads to impaired oocyte activation (Tesarik et al. 1998). This could be owing to a low concentration, or absence, of the sperm cytosolic factor (Palermo et al. 1997, Aslam et al. 1998), or due to the impermeability of the round-spermatid membrane inhibiting the release of sperm factor (Palermo et al. 1996) responsible for oocyte activation (Swann 1990). Saunders et al.(2002) proposed that the sperm-specific phospholipase- is the elusive sperm factor. In somatic cells, phospholipase generates InsP₃ (Berridge 1993) and is likely to do so in the oocyte. Calcium oscillations within oocytes are attributed to InsP₃ activity (Parrington et al. 1998, Halet et al. 2003). Hence, strontium, which promotes calcium oscillations through InsP₃ receptors in oocytes (Zhang et al. 2005), compensates for the lack of cytosolic factor activity in round spermatids.

Kimura & Yanagimachi (1995) demonstrated that superior outcomes could be achieved by injecting round spermatids into an activated oocyte at the telophase stage of the cell cycle. It has been demonstrated that round spermatids achieve the highest fertilization rates when the oocyte is activated 1 h before ROSI (Kimura & Yanagimachi 1995). In the present study, a means to accommodate this information, while still adhering to the 3-h strontium protocol was explored through various protocols. We hypothesized that conducting ROSI before second polar-body extrusion may have unwittingly inflicted meiotic-spindle disturbance, which has been previously demonstrated to be damaging to oocytes development after fertilization (Hardarson et al. 2000). Although it has been identified that mouse oocytes maintain the ability to result in a normal embryo if activated 60–80 min before and after ROSI (Kishigami et al. 2004) in the present study, activation of oocytes before introducing the round spermatid into the cytoplasm was superior to all other protocols. This resulted in a pup rate not significantly different than ICSI. In addition, the offspring obtained from ROSI were fertile as already identified by others (Tamashiro et al. 1999). The present study also indicates the importance of a competent oocyte activation protocol to promote development outcomes from round spermatids that are equivalent to those from mature spermatozoa. As clinical ROSI has been largely abandoned due to safety concerns, animal models such as used here may provide valuable insights. The present research suggests that clinical ROSI may benefit from the application of an optimal oocyte-activation technique that needs to be explored, particularly one that promotes calcium oscillations in human oocytes with no toxic effect on pre- and post-implantation development.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 20 July 2005
First decision 26 August 2005
Revised manuscript received 6 September 2005
Accepted 26 September 2005

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