The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.

INTRODUCTION

Traditionally, in nuclear transfer micromanipulation, the blind enucleation and the 3 o'clock position of the sperm injection rely on the MII (second meiotic metaphase) spindle being in close proximity to the FPB (first polar body). Provided care is taken to orient the polar body away from the site of injection, injection of sperm into ooplasm may not result in damage to the meiotic spindle [1]. In mammalian oocytes, however, the FPB does not accurately predict the location of the MII meiotic spindle. Blind removal of the cytoplasm beneath the FPB does not always allow for the removal of all the chromosomes because between 10% [2,3] and 25% [4] or more [5] of such oocytes still contain residual DNA. A portion of zygotes do not cleave or cleave abnormally after ICSI (intracytoplasmic sperm injection) [6]. The timing and position of ROSI (round spermatid injection) can also significantly influence pronuclei formation [7]. Furthermore, a high degree of misalignment between the meiotic spindle and the FPB is associated with an increased risk of fertilization abnormalities [8]. Although experiments using a polarized light microscope have shown that the FPB position cannot accurately predict the location of the meiotic spindle in the 15 h post hCG (human chorionic gonadotropin) hamster oocytes [9], the spatial relationship between the MII spindle and the FPB position needs further investigation in hamster oocytes of different ages. In addition, the polar body morphology was found to be associated with subsequent fertilization rate and embryonic quality of ICSI embryos [10], necessitating a study on FPB degeneration.

The golden hamster is an excellent experimental model. It represents an attractive species for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, reciprocal changes in the position of the FPB and chromosome set of golden hamster oocytes were examined under different conditions. We also investigated the timing of FPB degeneration and analysed the effect of colchicine or CB (cytochalasin B) treatment.

MATERIALS AND METHODS

Chemicals and animals

All chemicals were purchased from Sigma (St Louis, MO, U.S.A.) and Invitrogen (Grand Island, NY, U.S.A.) unless otherwise noted. The golden hamsters (females, 6 weeks old) were purchased from Changchun Hi-Tech Laboratory Animal Research Center (Changchun, China) and housed with three animals per cage under controlled light cycle (14 h light/10 h dark) conditions. Animal treatment complied with a protocol approved by the Jilin University Institutional Animal Care and Use Committee.

Collection of oocytes

In vivo maturation of oocytes derived from the oviducts of superovulated females

The hamsters were superovulated with PMSG [pregnant mare serum gonadotropin; 30 IU (international units), intraperitoneal] followed by hCG (30 IU; intraperitoneal) at 72 h intervals. Both PMSG and hCG used in the present study were from Ningbo Hormone Product (Ningbo, China). The superovulated hamsters were killed at different times (13.5, 18 and 23 h) after hCG injection, and the oviductal ampullae were collected and broken to release the COCs (cumulus–oocyte complexes). Then, cumulus cells were removed from COCs by pipetting with a thin pipette in M2 [11] containing 0.1% hyaluronidase (H4272; Sigma).

In vitro culture of oocytes derived from the oviducts of superovulated females

The superovulated hamsters were killed at 13.5 h after hCG injection. Then, the oocytes, with or without cumulus cells, were cultured in HECM-3 (hamster embryo culture medium-3) [12] for 5 and 10 h. After in vitro culture, the COCs were denuded by the addition of 0.1% hyaluronidase.

In vitro maturation of oocytes derived from the ovaries of PMSG-stimulated females

Female hamsters were killed 72 h after PMSG administration. The small vesicular follicles (0.5–1 mm in diameter) on the ovarian surface were incised with a scalpel in a Petri dish containing M2 to release the COCs. The COCs were cultured in 100 μl of microdrops of HECM-3 supplemented with 5 μg/ml of FSH (follicle-stimulating hormone; F2293; Sigma) and 5 μg/ml of LH (luteotrophic hormone; L5269; Sigma) for 18 and 23 h. Each microdrop contained 15 COCs. After in vitro culture, the COCs were denuded by the addition of 0.1% hyaluronidase.

Treatment of collected oocytes

Examination of FPB position in relation to the chromosome set of MII oocyte

Denuded oocytes from the above three collections were stained with 10 μg/ml of Hoechst 33342 (B2261; Sigma) for 5 min and evaluated under an inverted fluorescence microscope (Nikon). During observation, oocytes were rotated and positioned with micromanipulators to orientate the FPB at the 12 o'clock position and the chromosome in the same focal plane as the FPB. The location of the FPB relative to the MII spindle was recorded as the angle (0°–30°, 30°–90°, 90°–180°; Figures 1A–1F) between the line from the FPB to the centre of the oocyte and the line from the chromosome to the centre of the oocyte.

Changes in FPB positions and morphology in normal and colchicine-treated oocytes
Figure 1
Changes in FPB positions and morphology in normal and colchicine-treated oocytes

(A–H) Golden hamster oocytes collected at 18 h post hCG injection showing changes in FPB positions and morphology relative to the MII chromosome. The oocytes were stained with Hoechst 33342 and propidium iodide, examined with a PlanApo ×20/0.45 NA objective lens by phase-contrast and fluorescence microscopy (TE2000-U; Nikon) and processed by using MetaMorph software. (A, B) FPB in the 0°–30° zone; (C, D) FPB in the 30°–90° zone; (E, F) FPB in the 90°–180° zone; (G, H) FPB that had degenerated. (I–L) Golden hamster oocytes treated with colchicine. (I, J) Oocyte with a cytoplasmic protrusion (arrow) under phase-contrast and fluorescence microscopy with a PlanApo ×20/0.45 NA objective lens; (K, L) the spindle and chromosomes (arrow) in the protrusion under a confocal laser scanning microscope (FV1000; Olympus) and processed by FluoView software; the pictures were observed with a PlanApo ×100/1.4 NA oil immersion objective lens (temperature: 37.5°C).

Figure 1
Changes in FPB positions and morphology in normal and colchicine-treated oocytes

(A–H) Golden hamster oocytes collected at 18 h post hCG injection showing changes in FPB positions and morphology relative to the MII chromosome. The oocytes were stained with Hoechst 33342 and propidium iodide, examined with a PlanApo ×20/0.45 NA objective lens by phase-contrast and fluorescence microscopy (TE2000-U; Nikon) and processed by using MetaMorph software. (A, B) FPB in the 0°–30° zone; (C, D) FPB in the 30°–90° zone; (E, F) FPB in the 90°–180° zone; (G, H) FPB that had degenerated. (I–L) Golden hamster oocytes treated with colchicine. (I, J) Oocyte with a cytoplasmic protrusion (arrow) under phase-contrast and fluorescence microscopy with a PlanApo ×20/0.45 NA objective lens; (K, L) the spindle and chromosomes (arrow) in the protrusion under a confocal laser scanning microscope (FV1000; Olympus) and processed by FluoView software; the pictures were observed with a PlanApo ×100/1.4 NA oil immersion objective lens (temperature: 37.5°C).

Identification of degenerated FPBs

Three categories of analysed oocytes were stained with 10 μg/ml of propidium iodide (P4170; Sigma) for 10 min before observation. The FPB that were stained with propidium iodide were considered to have degenerated (Figures 1G and 1H).

CB and/or colchicine treatment

Denuded oocytes collected from superovulated hamster oviducts at 13.5 h post hCG were treated with 5 μg/ml of CB (C6762; Sigma) and 10 μg/ml of colchicine (C9754; Sigma) either individually or in combination for 5 h in HECM-3. The FPB positions were observed at the end of treatment to test whether the two chemicals had any effects. The oocytes in the control group were treated for the same period in HECM-3 with neither CB nor colchicine.

Examination of FPB, spindle and chromosomes of oocytes treated with CB and/or colchicine

Oocytes treated with CB and/or colchicine were collected and fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 40 min at room temperature (25°C). Fixed oocytes were permeabilized by transferring to PBS supplemented with 0.1% (w/v) Triton X-100 and 0.3% (w/v) BSA for 30–40 min at room temperature. After washing twice with PBS containing 0.01% Triton X-100, oocytes were incubated in a blocking solution (PBS containing 1% BSA) for 1 h at room temperature. The microtubules were localized by an incubation for 1 h at room temperature with an FITC-labelled mouse monoclonal antibody against α-tubulin (F2168; Sigma), which was diluted 1:100 in a blocking solution. Nuclear status of the oocytes was evaluated by staining with 10 μg/ml of propidium iodide in PBS for 10 min. After extensive washing, oocytes were mounted on slides with anti-fade medium [DABCO (1,4-diazadicyclo[2.2.2]octane); D-2522; Sigma]. Finally, the oocytes were observed on a confocal laser scanning microscope (Olympus).

Statistical analysis

Three replicate trials were conducted for each treatment. The mean percentage (±S.E.M.) was calculated for each experimental group. Data were analysed by one-way ANOVA by using SPSS (Statistics Production for Service Solution) software (version 12.0; SPSS Inc., San Rafael, CA, U.S.A.) after being transformed via LSD (least significant difference). The difference was considered statistically significant when P<0.05.

RESULTS

In vivo-matured oocytes collected from oviducts at different times post hCG injection were examined for the position of the FPB in relation to the MII oocyte chromosome. When oocytes were collected at 13.5 h post hCG, most of the FPBs were found in the 0°–30° zone. In contrast, a significantly lower percentage of FPBs were found in this range when oocytes were collected at 18 and 23 h post hCG (P<0.05), whereas more FPBs of 18 h/23 h post hCG oocytes were located in the zone of 30°–90° and 90°–180°. The percentage of FPBs stained by propidium iodide increased from 10 to 71% when oocytes were collected between 13.5 and 23 h, indicating that oocytes gradually degenerated in vivo with age. FPBs were not seen in some oocytes collected at 18 and 23 h post hCG injection (see Table 1 and Figures 1A–1H).

Table 1
Changes in the position of the FPB in in vivo-matured oocytes

Values with different superscript letters within the same column are significantly different (P<0.05).

0°–30°30°–90°90°–180°Degenerated FPBOocytes without FPB
Time post HCG (h)Total number of oocytes observedNumber%Number%Number%Number%Number%
13.5 73 60 82.10±3.47a 13 17.98±3.47a 0.00±0.00a 9.56±6.59a 0.00±0.00a 
18 50 24 46.32±7.82b 18.41±3.69a 13 26.63±5.47b 27 54.11±3.97b 8.64±4.54a 
23 82 28 33.33±7.69b 36 44.81±8.66b 17 20.74±0.74b 58 71.01±2.71c 2.44±2.14a 
0°–30°30°–90°90°–180°Degenerated FPBOocytes without FPB
Time post HCG (h)Total number of oocytes observedNumber%Number%Number%Number%Number%
13.5 73 60 82.10±3.47a 13 17.98±3.47a 0.00±0.00a 9.56±6.59a 0.00±0.00a 
18 50 24 46.32±7.82b 18.41±3.69a 13 26.63±5.47b 27 54.11±3.97b 8.64±4.54a 
23 82 28 33.33±7.69b 36 44.81±8.66b 17 20.74±0.74b 58 71.01±2.71c 2.44±2.14a 

The in vivo-matured oocytes (denuded and intact – with cumulus cells) collected at 13.5 h post hCG injection were cultured in vitro for 5 or 10 h and examined for FPB position. When denuded oocytes were cultured in vitro for 5 h, more FPBs were located in the 0°–30° zone, which is significantly higher than those oocytes cultured with cumulus (P<0.05). When denuded and intact oocytes were cultured in vitro for 10 h, more FPBs were located in the 90°–180° zone than those cultured in vitro for 5 h. Denuded oocytes cultured in vitro for 5 and 10 h were found to have a higher percentage of degenerated FPBs than intact oocytes, but this difference was not significant (see Table 2).

Table 2
Changes in the position of the FPB in in vitro-cultured oocytes

Values with different superscript letters within the same column are significantly different (P<0.05).

0°–30°30°–90°90°–180°Degenerated FPB
Culture duration (h)Total number of oocytes observedNumber%Number%Number%Number%
Without cumulus cells 72 31 43.05±0.71a 29 40.27±0.75a 12 16.68±0.40a 32 44.73±8.29a 
 10 63 24 35.87±9.65ab 20 34.11±13.00a 19 30.02±3.96b 43 62.80±19.43a 
With cumulus cells 46 10 24.32±7.86b 25 53.54±9.66a 11 22.14±4.37ab 13 32.11±7.29a 
 10 46 14 30.55±2.78ab 21 42.36±4.86a 11 27.08±5.24ab 23 45.14±7.25a 
0°–30°30°–90°90°–180°Degenerated FPB
Culture duration (h)Total number of oocytes observedNumber%Number%Number%Number%
Without cumulus cells 72 31 43.05±0.71a 29 40.27±0.75a 12 16.68±0.40a 32 44.73±8.29a 
 10 63 24 35.87±9.65ab 20 34.11±13.00a 19 30.02±3.96b 43 62.80±19.43a 
With cumulus cells 46 10 24.32±7.86b 25 53.54±9.66a 11 22.14±4.37ab 13 32.11±7.29a 
 10 46 14 30.55±2.78ab 21 42.36±4.86a 11 27.08±5.24ab 23 45.14±7.25a 

COCs were collected from small vesicular follicles on the ovarian surface at 72 h after PMSG injection in hamsters. These COCs matured in vitro for 18 and 23 h and were examined for FPB position. More FPBs were located in the 0°–30° zone when oocytes matured in vitro for 18 h than when oocytes matured in vitro for 23 h (P<0.05). The percentage of FPBs in the 30°–90° zone when oocytes matured in vitro for 18 h was significantly lower (P<0.05) than the percentage of FPBs when oocytes matured in vitro for 23 h (see Table 3).

Table 3
Changes in the position of the FPB in in vitro-matured oocytes

Values with different superscript letters within the same column are significantly different (P<0.05).

0°–30°30°–90°90°–180°Degenerated FPB
Culture duration (h)Total number of oocytes observedNumber%Number%Number%Number%
18 36 28 77.94±3.81a 10.85±6.19a 8.65±1.24a 0.00±0.00a 
23 36 14 38.83±1.54b 20 56.04±5.18b 5.13±5.13a 2.78±2.78a 
0°–30°30°–90°90°–180°Degenerated FPB
Culture duration (h)Total number of oocytes observedNumber%Number%Number%Number%
18 36 28 77.94±3.81a 10.85±6.19a 8.65±1.24a 0.00±0.00a 
23 36 14 38.83±1.54b 20 56.04±5.18b 5.13±5.13a 2.78±2.78a 

Reciprocal position of FPB to the MII oocyte chromosome was examined in oocytes collected at 13.5 h post hCG and treated with CB and colchicines for 5 h either individually or in combination. There was no significant difference in the percentage of FPBs observed in the 0°–30°, 30°–90° and 90°–180° zones between oocytes treated with CB and control oocytes; however, when oocytes were treated with colchicine, the percentage of FPBs located in these zones was significantly different from that in the control oocytes. Moreover, after treatment of oocytes with colchicine, 79.44% showed induced cytoplasmic protrusions, in which chromosomes were compartmentalized in budding polar bodies (see Figures 1I–1L). After treatment of oocytes with both CB and colchicine, the percentages of FPBs located in the 0°–30°, 30°–90° and 90°–180° zones were similar to those of oocytes treated with colchicine alone (see Table 4).

Table 4
Changes in the position of the FPB in oocytes treated with CB and/or colchicines

Values with different superscript letters within the same column are significantly different (P<0.05).

0°–30°30°–90°90°–180°Oocytes with cytoplasmic protrusion
TreatmentTotal number of oocytes observedNumber%Number%Number%Number%
Control 52 21 39.56±3.28a 23 46.04±6.17a 14.39±3.06a 0.00±0.00a 
CB 49 13 25.90±7.98a 26 54.51±14.20a 13.71±4.46a 0.00±0.00a 
Colchicine 43 11.04±3.32b 7.14±7.14b 2.38±2.38b 34 79.44±8.04b 
CB+colchicine 41 18.56±10.83a 10.23±5.37b 5.41±2.76ab 27 65.80±13.59b 
0°–30°30°–90°90°–180°Oocytes with cytoplasmic protrusion
TreatmentTotal number of oocytes observedNumber%Number%Number%Number%
Control 52 21 39.56±3.28a 23 46.04±6.17a 14.39±3.06a 0.00±0.00a 
CB 49 13 25.90±7.98a 26 54.51±14.20a 13.71±4.46a 0.00±0.00a 
Colchicine 43 11.04±3.32b 7.14±7.14b 2.38±2.38b 34 79.44±8.04b 
CB+colchicine 41 18.56±10.83a 10.23±5.37b 5.41±2.76ab 27 65.80±13.59b 

DISCUSSION

SCNT (somatic cell nuclear transfer), ICSI and ROSI procedures rely on knowledge of the spatial relationship between the MII oocyte chromosome and the FPB. Although recent observations have shown that the position of the FPB does not accurately predict the location of the meiotic spindle and chromosome set in MII oocytes of monkey [13], human [14] and mouse [15], a detailed study on the change in the FPB position and the factors affecting it is lacking. Silva et al. [9] have studied the spatial relationship between the MII spindle and the FPB in a polarized light microscope [PolScope (Carl Zeiss)] and found that, in 30 oocytes from 12 hamsters, the FPB position did not accurately predict the location of the meiotic spindle. We conducted this study in order to characterize changes in the FPB position relative to the MII oocyte chromosome of hamsters as well as the timing of FPB degeneration in detail; we also examined chemicals (CB and colchicine) that affect FPB deviation.

When in vivo-matured oocytes collected from oviducts at different times post hCG injection were examined for the changes in FPB position, 82.1% of oocytes collected at 13.5 h post hCG had the FPB adjacent to the MII oocyte chromosome (within the 0°–30° zone). The position of the FPB was different in oocytes collected at 18 and 23 h post hCG, with fewer FPBs in the 0°–30° zone and more FPBs in the 30°–90° and 90°–180° zones. It was suggested that this deviation was caused by removal of the cumulus cells [8]. The position of the FPBs was not significantly different between the groups when ovulated oocytes, denuded and intact, were cultured in vitro. This may indicate that cumulus cell removal does not affect the FPB position.

Compared with in vivo-matured oocytes, oocytes matured in vitro for 18 and 23 h exhibited more FPBs in the 0°–30° zone and fewer in the 90°–180° zone, suggesting that oocytes that mature in vitro age slower than those matured in vivo. Hardarson et al. [14] and Rienzi et al. [8] obtained similar results in human oocytes, finding that in vivo-matured oocytes have a significantly larger deviation of the MII spindle from the FPB when compared with in vitro-matured oocytes. Moon et al. [16] proposed that the different spindle positions observed in in vivo- and in vitro-matured oocytes may reflect differences in their cytoplasmic maturation processes.

It has been reported that FPB morphology reflects oocyte competence. Oocytes with an intact normal-sized FPB are thought to generate better embryos, higher blastocyst yield and increased pregnancy and implantation rates [17]. By evaluating FPB morphology (intact, degeneration) in combination with propidium iodide staining, we showed that more in vivo-matured oocytes degenerated as the oocytes aged. At ovulation, 9.6% of FPBs degenerated (13.5 h post hCG); 71% of FPBs degenerated when the oocytes were collected at 23 h post hCG injection; a few FPBs of the older oocytes (18 and 23 h post hCG) disappeared in vivo. Compared with the mouse [15], FPBs of hamster oocytes degenerate more slowly. In mice, 19.5% of FPBs begin to degenerate at ovulation (12 h post hCG injection), and by 20 h post hCG injection the proportion of degenerated FPB increases to 100%; by 32 h post hCG injection, only 9.4% of oocytes have FPBs. When ovulated hamster oocytes, denuded and intact, were cultured in vitro for 5 h (18 h post hCG) or 10 h (23 h post hCG), more denuded oocytes degenerated than those cultured with cumulus cells. When the COCs matured in vitro for 18 and 23 h, the oocytes aged more slowly than those matured in vivo and cultured in vitro. It is reported that the rate of FPB degeneration is species dependent. Evsikov and Evsikov [18] found that more than half of the FPBs of mouse oocytes degenerate within a few hours after ovulation; however, in human oocytes, many FPBs persist for more than 20 h after ovulation [19]. Although degeneration of polar bodies is likely to be an apoptotic process [20,21], the factors that determine the individual and species differences in the degeneration rates of polar bodies are not well understood.

It has been reported that CB and colchicine affect the oocyte cytoskeleton [15]. In the present study, when newly ovulated hamster oocytes were treated with CB, the portion of FPBs located in the 0°–30°, 30°–90° and 90°–180° zones was not significantly different from that in the control oocytes. This may indicate that the cytoskeleton may not be involved in FPB deviation after ovulation. When oocytes were treated with colchicine, however, the portion of FPBs located in the same zones was significantly different from that in the control oocytes. Moreover, 79.44% of oocytes treated with colchicine showed induced cytoplasmic protrusions in which chromosomes were compartmentalized in budding polar bodies; this result is different from that obtained in mouse oocytes [15]. Although cytoplasmic protrusions do not mean complete enucleation, they assisted enucleation by micromanipulation. Until now, demecolcine [22], etoposide [23] and cycloheximide [24] have been used to induce enucleation. Some animals have been derived from the activated cytoplasts prepared by induced enucleation [22,25]. There has not yet been a report on using colchicine to induce enucleation, so further investigation is needed.

Comparing the three regimes of oocyte collection and chemical treatment, the oocytes collected directly from oviducts 13.5 h post hCG injection showed much better orientation, as this category of oocytes were in vivo-matured and easy to collect, and most FPBs (82.1%) oriented the position of the oocyte chromosome set and spindle. Most oocytes (77.94%) matured in vitro for 18 h showed better orientation of the reciprocal position of the FPB and oocyte chromosome set. The colchicine could induce the chromosome set and spindle of most oocytes (79.44%) to compartmentalize in protrusions that are easy to be removed, so the colchicine was a good choice to assist enucleation. Further research is necessary to test which source of oocytes is the best for embryo reconstruction and developmental potential.

In conclusion, the results of the present study demonstrate that the change in FPB position in relation to the MII oocyte chromosome set is increasing with age in in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. Colchicine induced cytoplasmic protrusions in hamster oocytes, whereas CB almost has no effect on them. These results characterize changes in FPB position in relation to the MII oocyte chromosome set under various culture conditions. Freshly ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.

Abbreviations

     
  • CB

    cytochalasin B

  •  
  • COC

    cumulus–oocyte complex

  •  
  • FPB

    first polar body

  •  
  • hCG

    human chorionic gonadotropin

  •  
  • HECM-3

    hamster embryo culture medium-3

  •  
  • ICSI

    intracytoplasmic sperm injection

  •  
  • IU

    international unit

  •  
  • MII

    second meiotic metaphase

  •  
  • PMSG

    pregnant mare serum gonadotropin

  •  
  • ROSI

    round spermatid injection.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant number 30671510]; and the Jilin University Research Starting Fund of China.

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