- Email: [email protected]
Effect of sugars on maturation rate of vitrified-thawed immature porcine oocytes
Animal Reproduction Science 106 (2008) 25–35
Effect of sugars on maturation rate of vitrified-thawed immature porcine oocytes Jing Huang a , Qingwang Li a,b,∗ , Rui Zhao b , Wenye Li a , Zengsheng Han b , Xiaoyu Chen c , Bo Xiao a,d , Shuyun Wu a , Zhongliang Jiang a , Jianhong Hu a , Lei Liu b a
b
College of Animal Science, Northwest Agriculture and Forestry University, Yangling, Shannxi Province 712100, China Department of Biological Engineering, College of Enviroment and Chemical Engineering, Yanshan University, Qinhuangdao, Hebei Province 066004, China c Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province 310021, China d College of Life Sciences, LuDong University, Yantai, Shandong Province 264025, China Received 3 October 2006; received in revised form 21 December 2006; accepted 23 March 2007 Available online 30 March 2007
Abstract This study examined the effects of monosaccharide (glucose), disaccharide (sucrose) and polysaccharides (Ficoll and Lycium barbarum polysaccharide (LBP)) at different concentrations, using ethylene glycol (EG) as membrane-permeating cryoprotectant, on in vitro maturation of vitrified-thawed immature (GV) porcine oocytes. A total of 1145 oocytes were obtained by follicle aspiration from 496 ovaries of pigs slaughtered at a local abattoir and vitrified using a five-step method. After thawing and removal of cryoprotectant, oocytes were cultured for 44 h at 39 ◦ C in a humidified atmosphere of 5% CO2 in air. Oocytes were stained with DAPI and nuclear maturation was examined. The highest maturation rates were obtained in 1.5 M glucose (8.62%), 0.75 M sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.10 g/ml LBP (20.69%), respectively. The maturation rate using 0.75 M sucrose or 0.10 g/ml LBP was significantly higher compared to 1.5 M glucose (P < 0.05), but there was no significant difference from using 3.0 g/ml Ficoll (P > 0.05). The percentage of oocytes reaching metaphase II (MII) stage in the cryopreserved groups was significantly lower than control (P < 0.05). These results suggest that LBP is an effective non-permeating membrane cryoprotectant and 0.75 M sucrose or 0.10 g/ml LBP can be used as the vitrification solution for immature porcine oocytes. © 2007 Elsevier B.V. All rights reserved. Keywords: Porcine; Oocyte; Vitrification; Maturation ∗ Corresponding author. Present address: College of Animal Science, Northwest Agriculture and Forestry University, Yangling, Shannxi Province 712100, China. Tel.: +86 335 8074662; fax: +86 335 8074662. E-mail address: [email protected] (Q. Li).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.03.023
26
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
1. Introduction The rapid development of reproductive technologies, such as in vitro maturation, in vitro fertilization and in vitro embryo production has lead to increased demand for mammalian oocytes. Therefore, it would be a significant advance for basic research and commercial applications (Schroeder et al., 1990; Candy et al., 1994), if immature oocytes could be cryopreserved before maturation and use. Cryopreservation of immature oocytes has been reported with varying degrees of success in several mammalian species, such as mice (Eroglu et al., 1998; Moffa et al., 2002), cattle (Suzuki et al., 1996), buffalo (Wani et al., 2004) and humans (Tucker et al., 1998; Isachenko et al., 2006). However, immature porcine oocytes remain one of the most difficult to successfully cryopreserve due to their sensitivity to cooling and freezing. Many studies have showed that the in vitro maturation rate after vitrification is extremely low (Park et al., 2005; Rojas et al., 2004; Hara et al., 2005). Little success has been achieved with cryopreserving immature porcine oocytes. Isachenko et al. (1998) obtained 22% metaphase II (MII) stage oocytes after vitrification of immature (GV) oocytes by pretreating with cytochalasin B (CB). Fujihira et al. (2004) improved the maturation rate to 37.1% after vitrification of GV oocytes using a cryotop sheet. There are many studies involving cryoprotectants, but the role of sugars in the vitrification solution for immature (GV) porcine oocytes cryopreservation has not been evaluated. The protective action of sugars is very complex, attributable to a number of their special properties (Fabbri et al., 2000). Due to their large molecular size, sugars could cause an osmotic gradient across the cell membrane, which enhance dehydration of the cell before freezing of extracellular water. In addition, it is suggested that sugars are capable of preserving the structural and functional integrity of cell membranes at low water activities (Hotamisligil et al., 1996). As early as 1993, mouse embryos were vitrified with 2.75 M dimethyl sulfoxide and 2.75 M propylene glycol supplemented with 1.0 M sucrose after a 0.5-min exposure, which exhibited a significantly higher in vitro survival rate (82%) than the solution without 1.0 M sucrose (44%) (Tada et al., 1993). Furthermore, it has been reported that vitrification solution with added sugars could significantly improve the survival rate of the vitrified bovine blastocysts (Saito et al., 1994). Toth et al. (1994) also showed that the maturation rate of frozen human immature oocytes was improved when sugars were added. In a study of common carp embryos, it was clearly suggested that sugars lowered the toxicity of permeating cryoprotectants (Ahammad et al., 2002). In earlier studies, many commercial sugars were used widely as cryoprotectants, while over the last few decades, the biological activities of plant polysaccharides have attracted increased attention in biochemistry and medicine. However, to date, there is no report on the effects of plant polysaccharides as a cryoprotectant on the vitrification of immature porcine oocytes. The purpose of this study was to investigate the effects of monosaccharide (glucose), disaccharide (sucrose) and polysaccharides (Ficoll and LBP) at different concentrations on the post-thaw maturation rates of porcine oocytes and evaluate sugars as potential cryoprotectants for vitrification of porcine immature oocytes. To the best of our knowledge, this is the first report on the effect of Lycium barbarum polysaccharide on the vitrification of porcine immature oocytes. 2. Materials and methods Unless otherwise stated, all the chemicals used in this study were purchased from Sigma (St. Louis, MO, USA).
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
27
2.1. Preparation of L. barbarum polysaccharide Dried L. barbarum fruits were ground to a powder and the powder refluxed twice with petroleum ether (each time for 1 h) to remove lipids (Tianjin, China), then refluxed again with 80% ethanol (Tianjin, China) twice (each time for 2 h) to remove monosaccharides and oligosaccharides. The residue was extracted three times in hot water (90 ◦ C) and the combined filtrate concentrated through decompression using a rotary evaporator (Shanghai, China). A small amount of activated carbon (Qingdao, China) was added to remove color. After filtering, 95% ethanol was added to the filtrate and left overnight. The precipitate was washed sequentially using 95% ethanol, 100% ethanol, acetone and ether (Tianjin, China). Finally, the precipitate was collected and vacuum-dried to give the polysaccharide. 2.2. Ovaries selection and oocytes collection Ovaries were obtained from prepubertal pigs within 5 min after slaughtered at a local abattoir. The selected ovaries were in the follicular phase with no apparent corpus lutea and were transported to the laboratory in D-PBS containing 150 IU/ml penicillin and 100 g/ml streptomycin at 35 ◦ C within 2 h. The cumulus–oocyte complexes (COCs) were collected from non-atretic follicles (2–6 mm in diameter) by aspiration with an 18G needle attached to a 10-ml sterile syringe. Only oocytes with compact cumulus cells showing a homogeneous cytoplasm were selected and rinsed three times in TL–HEPES–PVA medium, as described previously (Funahashi et al., 1997). 2.3. Vitrification and thawing As shown in Table 1, the vitrification solutions were prepared using a holding medium (TCM199–Hepes supplemented with 20% fetal calf serum (FCS, Hyclone)). As ethylene glycol (EG) has low toxicity and good permeating capability, it was used as the cryoprotectant in this experiment. All manipulations were performed on a 41 ◦ C hot-plate in a room at 25–27 ◦ C to maintain the media at 38.5–39 ◦ C. Oocytes were first equilibrated in the holding medium for 5 min and then treated with 7.5 g/ml cytochalasin B for 25 min at 38.5 ◦ C. Oocytes were exposed to 5, 10, 20, 30 and 40% ethylene glycol, which corresponded to a 12.5, 25, 50, 75 and 100% sugar concentration of each treatment group for 3, 3, 2, 1 and 0.5 min, respectively (Table 1). Each group of 5–8 oocytes was loaded into 0.25 ml straws containing a 0.5-cm column of vitrification solution (40% EG), which were heat-sealed and immediately dipped in liquid nitrogen. After being stored in liquid nitrogen for at least 1 week, the straw was thawed in a water bath at 37 ◦ C for 5 s. The COCs were immediately expelled into a Petri dish with 2 ml of 0.75 M sucrose solution for 5 min. The oocytes were then transferred to the decreasing concentrations of 0.5 and 0.25 M sucrose Table 1 The component of vitrification solution and exposed time Steps
First step
Second step
Third step
Fourth step
Fifth step
Vitrification solution
5% EG (v/v) + 12.5% × A (M)
40% EG (v/v) + 100% × A (M)
3
20% EG (v/v) + 50% × A (M) 2
30% EG (v/v) + 75% × A (M)
Treat time (min)
10% EG (v/v) + 25% × A (M) 3
1
0.5
(EG) ethylene glycol and (A) the different sugar concentrations of treatment groups.
28
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
at 2-min intervals. The oocytes were subsequently washed three times in maturation medium, which was modified TCM-199 (TCM199; Gibco, Grand Island, NY, USA; Cat. No. 31100-035) supplemented with 0.1% PVA (Cat. No. P8136), 3.05 mM glucose (Ameresco, Cat. No. 0188), 0.91 mM sodium pyruvate (Cat. No. P5280), 0.57 mM/ml l-cysteine (Cat. No. C7352), 10 IU/ml PMSG (Cat. No. G4877), 10 IU/ml hCG (Cat. No. C1063), 10 ng/ml EGF (Promega, Cat. No. G5021), 50 IU/ml penicillin G and 50 IU/ml streptomycin sulfate. The morphological appearance of the oocytes was evaluated after thawing under an inverted microscope. The oocytes with spherical and symmetrical shape and no signs of lyses/degeneration were considered normal, whereas oocytes with ruptured zona pellucida, fragmented cytoplasm or degenerative signs were classified as abnormal. 2.4. In vitro maturation of the vitrified-thawed oocytes The washed COCs were kept in the oocyte maturation medium for 20 min in a CO2 incubator and then transferred to 500 l of the maturation medium in a four-well culture plate. The COCs were cultured for 44 h at 39 ◦ C in a humidified atmosphere of 5% CO2 in air. Fresh oocytes were also cultured in the same manner as control. 2.5. Assessment of nuclear maturation After a 44-h incubation in the maturation medium, oocytes with granulated ooplasm, broken membrane and zona pellucida were recorded as degenerated. Cumulus cells were removed by mixing oocytes with 0.1% hyaluronidase in phosphate-buffered saline (PBS). The nuclear status of the denuded oocytes was determined by DAPI (Cat. No. D9542) staining. The oocytes were fixed in PBS with 2% (w/v) formaldehyde, washed with PBS, stained with 2.5% (w/v) DAPI and mounted on slides (Mori et al., 1988). The nuclear state of the stained oocytes was assessed under a fluorescence microscope (TS100, Nikon, Japan) at 450 nm. Oocytes with diffused or slightly condensed chromatin were classified as being in the GV stage. Oocytes that possessed clumped or strongly condensed chromatin forming an irregular network of individual bivalents (prometaphase) or a metaphase plate but no polar body were classified as being in the MI stage. Oocytes with either a polar body or two bright chromatin spots were classified as being in the MII stage (Fig. 1). 2.6. Statistical analysis Each experiment was repeated four times. All data of each stage of meiosis were subjected to one-way ANOVA using SPASS (version 11.0 for Windows). Values were compared by a Duncan test for multiple comparisons. A probability of P < 0.05 was considered to be statistically significant. 3. Results Our experimental results showed that oocytes displayed severe dehydration when incubated with vitrification solution but that most of them could revert to normal when removed from the cryprotectants. A total of 14 zona pellucide breakages were noticed, 6 in glucose, 3 in sucrose, 3 in Ficoll and 2 in LBP. The percentage of oocytes reaching metaphase II stage was lower (P < 0.05) in the cryopreserved groups compared to control and the expansion effect of cummlus cell in the
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
29
Fig. 1. Porcine oocytes stained with DAPI and visualized under UV light. Germinal vesicle (A), metaphase I (B) and metaphase II (C) (×400).
30
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
Table 2 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of glucose Glucose concentrations (M)
Total oocytesa
GVb (%)
MIc (%)
MIId (%)
Dege (%)
2.0 1.5 1.0 0.5 Control
63 58 53 62 59
43a (68.25) 37a (63.79) 36a (67.92) 44a (70.97) 3b (5.08)
10a (15.87) 10a (17.24) 9a (16.98) 5b (8.06) 5b (8.47)
3a (4.76) 5a (8.62) 3a (5.67) 3a (4.84) 47b (79.66)
7ab (11.11) 6ab (10.34) 5ab (9.43) 10b (16.13) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times. b GV: germinal vesicle. c MI: metaphase I. d MII: metaphase II. e Deg: degenerate.
control group was better than that in the cryopreserved groups. In addition, the maturation rate of oocytes was low in vitrification solutions supplemented with low sugar concentrations. After culture, the percentage in GV stage was not significantly different in the groups with different concentrations of glucose or Ficoll. However, in the sucrose and LBP groups, significantly more GV stage ooctyes were found in the low compared to the high concentration groups. Regarding the effect of glucose on the maturation rates of vitrified-thawed immature porcine oocytes, as shown in Table 2, the highest rate was obtained in 1.5 M glucose (8.62%), and it was not significantly different from oocytes vitrified in any other concentration (P > 0.05). The percentage of oocytes reaching metaphase I stage in 2.0 M (15.87%), 1.5 M (17.24%) or 1.0 M (16.98%) glucose was higher than that in 0.5 M (8.06%) glucose and control (8.47%) (P < 0.05). With respect to the effect of sucrose on maturation rates of vitrified-thawed immature porcine oocytes, the results in Table 3 show that the highest maturation rate was obtained in 0.75 M sucrose (20.0%, P < 0.05), but it was not significantly different from those vitrified in 1.0 M (17.65%) and 0.5 M (18.97%) sucrose (P > 0.05). The percentage of oocytes reaching metaphase I stage was not significantly different in the groups vitrified using 1.0 M (17.65%), 0.75 M (16.67%) or 0.5 M (12.07%) sucrose (P > 0.05). As shown in Table 4, the maturation rate of oocytes in the Ficoll group was similar to those in the glucose and sucrose groups with increasing concentrations. The highest maturation rate was observed in the group vitrified in 3.0 g/ml Ficoll (13.79%), but it was not significantly different from the other concentrations of Ficoll (P > 0.05).
Table 3 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of sucrose Sucrose concentrations (M)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
1.0 0.75 0.5 0.25 Control
51 60 58 60 59
27a (52.94) 32ab (53.33) 35b (60.34) 43c (71.67) 3d (5.08)
9a (17.65) 10ab (16.67) 7abc (12.07) 3c (5.0) 5bc (8.47)
9ab (17.65) 12b (20.0) 11ab (18.97) 5a (8.33) 47c (79.66)
6a (11.76) 6a (10.0) 5a (8.62) 9a (15.0) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times.
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
31
Table 4 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of ficoll Ficoll concentrations (g/ml)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
4.0 3.0 2.0 1.0 Control
60 58 50 61 59
40a (66.66) 37a (63.79) 35a (70.0) 39a (63.94) 3b (5.08)
8a (13.33) 7a (12.07) 4a (8.0) 7a (11.48) 5a (8.47)
7a (11.67) 8a (13.79) 3a (6.0) 4a (6.56) 47b (79.66)
5a (8.33) 6a (10.34) 8ab (16.0) 11b (18.03) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times. Table 5 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of L. barbarum polysaccharide L. barbarum polysaccharide concentrations (g/ml)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
0.10 0.075 0.05 0.025 Control
58 49 51 57 59
31a (53.45) 27a (55.10) 35ab (68.63) 39b (68.42) 3c (5.08)
10a (15.15) 8a (16.33) 5a (9.80) 7a (12.28) 5a (8.47)
12a (20.69) 9ab (18.37) 6b (11.76) 4b (7.02) 47c (79.66)
5a (8.62) 5a (10.20) 5a (9.80) 7a (12.28) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times.
Regarding the effect of LBP on maturation rates of vitrified-thawed immature porcine oocytes, the results in Table 5 show that the highest maturation rate was obtained in 0.10 g/ml LBP (20.69%), which was significantly higher than in 0.05 g/ml (11.76%, P < 0.05) and 0.025 g/ml (7.02%, P < 0.05), but it was not significantly different from those vitrified in 0.075 g/ml LBP (18.37%, P > 0.05). Furthermore, the expansion effect of cumulus cells in 0.10 and 0.075 g/ml LBP was superior compared to other cryopreserved groups. To assess the efficacy of sugars, four types were compared as to their effect on post-thaw oocyte maturation rates. The results are summarized in Fig. 2 and show that the highest maturation rate of vitrified-thawed porcine oocytes was obtained sequentially in 1.5 M glucose (8.62%), 0.75 M
Fig. 2. Maturation rates of vitrified-thawed immature porcine oocytes in vitrification solution adding 1.5 M glucose, 0.75 M sucrose, 3.0 g/ml Ficoll and 0.10 g/ml LBP. Values with different letters are significantly different (P < 0.05).
32
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.10 g/ml LBP (20.69%). The maturation rate of vitrified-thawed porcine oocytes using 1.5 M glucose was significantly lower than 0.75 M sucrose or 0.10 g/ml LBP (P < 0.05), but it was not significantly different from that in the 3.0 g/ml Ficoll added group (P > 0.05). 4. Discussion Cryoprotectants are divided into two types: membrane-permeating (e.g. ethylene glycol, dimethyl sulphoxide) and membrane non-permeating (e.g. sucrose, Ficoll). The membranepermeating cryoprotectants decrease the freezing point of the solution and prevent oocyte damage from high electrolyte concentrations (Rall et al., 1984). The membrane non-permeating cryoprotectants exerted their beneficial effects by cellular dehydration, caused by changing osmotic pressure. At present, the combined use of both cryoprotectants is standard. The present study attempted to evaluate the effect of varying levels of glucose, sucrose, Ficoll and LBP on the maturation rate of vitrified immature porcine oocytes. The experimental results showed that the best maturation rate was obtained by adding 0.10 g/ml LBP (20.69%), which is clearly higher than that reported by Rojas et al. (2004) in immature porcine oocytes (6.1%) vitrified in 40% EG + 0.5 M sucrose in open pulled straws (OPS). It is also higher than that reported by Park et al. (2005) in immature porcine oocytes (15.0%) vitrified after centrifugation and partial delipation. Vincent et al. (1991) suggested that sudden addition and removal of sucrose could result in significant zona pellucida hardening in MII stage mouse oocytes. In our study, the higher maturation rates of vitrified-thawed immature porcine oocytes may be related to the manner of exposure from low to high sugar concentrations. However, the maturation rates in this study were lower than those reported by Fujihira et al. (2004) for MII porcine oocytes (37.1%) employing the cryotop sheet and leading to rapid cooling rate of minimum volume. The maturation rate of vitrified-thawed immature porcine oocytes is lower than in other species (Wani et al., 2004; Moffa et al., 2002), the main reason being that immature porcine oocytes are very sensitive to cooling. Cytoskeleton elements in GV stage oocytes were straight and rigid, but in the MII stage they were undulating and flexible (Allworth and Albertini, 1993). In addition, due to high lipid levels in porcine oocytes, the hardened lipids produced by vitrification might also cause deformation and disruption of the cytoskeleton, leading to permanent damage (Orief et al., 2005). Sugars, as non-permeating cryoprotectants, have been used in the vitrification of oocytes or embryos of many species. The protective action of sugar is very complex and they could preserve the structural and functional integrity of the membrane at low water activities (Hotamisligil et al., 1996). Adding sugars to the vitrification solution could enhance viscosity, whereby incubation of cells in this solution before vitrification helps withdraw more water from the cells and reduce exposure of cells to the toxic effects of the cryoprotectants (Orief et al., 2005). Some reports have shown that the effect of added sugar was better than no sugar in the cryopreservation of human immature oocytes (Toth et al., 1994) and bovine blastocysts (Saito et al., 1994). Previous studies have demonstrated that monosaccharide was a more effective osmotic buffer than disaccharide (McWilliams et al., 1996). However, in our study, the maturation rate (8.62%) of vitrified-thawed immature porcine oocytes in the added glucose group was significantly lower than in the sucrose group (20.0%). It is hypothesized that osmotic stress in the vitrification solution supplemented with glucose was higher than that with sucrose, which could damage oocytes or that the temperature of vitrification in the solution with added sucrose was higher than the same concentration of added glucose (Kuleshova et al., 1999). In the present study, an oocyte maturation rate of 20.0%
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
33
was obtained using a medium containing 40% EG and 0.75 M sucrose, which was the highest of all four added sucrose groups and higher than that reported by Rojas et al. (2004) using the same vitrification solution. Polysaccharides could influence the viscosity of the vitrification solution and, thereby reduce the toxicity of the cryoprotectant through lowered concentration and prevent embryos from cryoinjury by reducing mechanical stress which occurs during cryopreservation (Dumoulin et al., 1994). Our observations show that the effect of Ficoll on the maturation rate of the vitrified-thawed immature porcine oocytes was not as good as with LBPs. Some reports have indicated that cryopreservation could damage antioxidant enzymes that protected cells against lipid peroxidation and freeze–thaw stress could be modified by incubating the embryo in the presence of inhibitors of membrane lipid peroxidation (Tarin and Trounson, 1993). LBP, extracted from the traditional Chinese herb L. barbarum, was found to have anticancer, antioxidant, hypoglycemic and immunological activities (Liu et al., 1996; Li et al., 1991; Zhao et al., 2005). LBP also protected seminiferous epithelium from structural damage and apoptosis in a testicular tissue culture system (Wang et al., 2002). Our experimental results suggest that adding LBP to vitrification solution might reduce the production of reactive oxygen species (ROS) and improve the antioxidant enzyme potential of oocytes, thereby preventing plasma membranes from lipid peroxidation and stabilizing membrane structure and permeability. LBP is a large polysaccharide molecule binding a protein, identified by ultraviolet, infrared spectroscopy and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Laemmli, 1970) and stained with Ag (Wray et al., 1981). Hays et al. (1996) reported that antifreeze glycoproteins (AFGPs) found in the blood of polar fish could inhibit leakage of intracellular contents across membranes during thermotropic phase transitions. O’Neil et al. (1998) reported that antifreeze glycoproteins could also be bound to the surface of growing ice-crystals, inhibiting the addition of any further water molecules. In this study, it was concluded that LBP exerted a similar effect to antifreeze glycoprotein. LBP also possesses enormous potential for structural variability, which could form a viscous matrix for encapsulation of the oocytes and preventing ice-crystal damage during cooling and warming. In this study, the maturation rates of vitrified-thawed immature porcine oocytes were superior in every group with added 1.5 M glucose (8.62%), 0.75 M sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.1 g/ml LBP (20.69%). In groups with added glucose, sucrose and Ficoll, the maturation rates of vitrified-thawed immature porcine oocytes were higher with increasing sugar concentrations; however, maturation rates decreased when sugar concentrations reached a certain level in the vitrification solution. An appropriate osmotic stress in the vitrification solution is an important factor for improving the maturation rate of vitrified-thawed immature oocytes, which could be regulated by sugars. Fabbri et al. (2001) demonstrated that increasing the sucrose concentration from 0.1 to 0.3 M in a freezing medium improved the survival rate of human oocytes after vitrification and prevented the formation of intracellular ice. On the other hand, Mullen et al. (2004) verified that increased sucrose concentration imposed a greater osmotic stress and increased the likelihood of damage to the spindle. A proper balance between oocyte dehydration and spindle damage is necessary, which are both caused by the osmotic stress of sugars. LBP was difficult to dissolve in the vitrification solution compared to other sugars, such as Ficoll; therefore, we believe that the highest maturation rate of vitrified-thawed immature porcine oocytes in the LBP group has not been reached. The maturation rate of vitrified porcine immature oocytes would be improved, if the concentration of LBP can be increased. In conclusion, our experimental results have demonstrated that porcine oocytes could be vitrified successfully by supplementation with 0.75 M sucrose or 0.1 g/ml LBP. Further research is needed, such as purification of LBP for higher solubility in the vitrification solution, to find a more effective non-permeating cryoprotectant.
34
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
Acknowledgement This research was supported by the Institutes of Science and Technology of Qin Huangdao City of Hebei Province of China (No. D08). References Ahammad, M.M., Bhattacharyya, D., Jana, B.B., 2002. The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures. Cryobiology 44, 114–121. Allworth, A.E., Albertini, D.F., 1993. Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton. Dev. Biol. 158, 101–112. Candy, C.J., Wood, M.J., Whittingham, D.G., Merriman, J.A., Choudhury, N., 1994. Cryopreservation of immature mouse oocytes. Hum. Reprod. 9, 1738–1742. Dumoulin, J.C., Bergers-Janssen, J.M., Pieters, M.H., Enginsu, M.E., Geraedts, J.P., Evers, J.L., 1994. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil. Steril. 62, 793–798. Eroglu, A., Toner, M., Leykin, L., Toth, T.L., 1998. Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes. J. Assist. Reprod. Genet. 15, 447–454. Fabbri, R., Porcu, E., Marsella, T., Primavera, M.R., Rocchetta, G., Ciotti, P.M., Magrini, O., Seracchioli, R., Venturoli, S., Flamigni, C., 2000. Technical aspects of oocyte cryopreservation. Mol. Cell. Endocrinol. 169, 39–42. Fabbri, R., Porcu, E., Marsella, T., Roccetta, G., Venturoli, S., Flamigni, C., 2001. Human oocytes cryopreservation: new perspectives regarding oocyte survival. Hum. Reprod. 16, 411–416. Fujihira, T., Kishida, R., Fukui, Y., 2004. Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants. Cryobiology 49, 286–290. Funahashi, H., Cantley, T.C., Day, B.N., 1997. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol. Reprod. 57, 49–53. Hara, K., Abe, Y., Kumada, N., Aono, N., Kobayashi, J., Matsumoto, H., Sasada, H., Sato, E., 2005. Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitriffication. Cryobiology 50, 216–222. Hays, L.M., Feeney, R.E., Crowe, L.M., Crowe, J.H., Oliver, A.E., 1996. Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions. Biophysics 93, 6835–6840. Hotamisligil, S., Toner, M., Powers, R.D., 1996. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol. Reprod. 55, 161–168. Isachenko, V., Montag, M., Isachenko, E., Dessole, S., Nawroth, F., van der Ven, H., 2006. Aseptic vitrification of human germinal vesicle oocytes using dimethyl sulfoxide as a cryoprotectant. Fertil. Steril. 85, 741–747. Isachenko, V., Soler, C., Isachenko, E., Perez-Sanchez, F., Grishchenko, V., 1998. Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant. Cryobiology 36, 250–253. Kuleshova, L.L., MacFarlane, D.R., Trounson, A.O., Shaw, J.M., 1999. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38, 119–130. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680–685. Li, W., Pai, S.Z., Ma, W., 1991. Kinetic observation on the change of SOD, LPO, Hb in blood of old people with the Barbary Wolfberry (Lycium barbarum). Chin. Tradit. Herbal Drugs 22, 251–268. Liu, J.L., Zhang, L.H., Qian, Y.K., 1996. Immune tumor-inhabition of Lycium barbarum polysaccharide on S180-bearing mice. Chin. J. Immunol. 12, 115–117. McWilliams, R.B., Gibbons, W.E., Leibo, S.P., 1996. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Cryobiology 54, 1059–1069. Moffa, F., Comoglio, F., Krey, L.C., Grifo, J.A., Revelli, A., Massorbrio, M., Zhang, J., 2002. Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes. Hum. Reprod. 17, 178–183. Mori, C., Hashimoto, H., Hoshino, K., 1988. Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice. Biol. Reprod. 39, 737–742. Mullen, S.F., Agca, Y., Broermann, D.C., Jenkins, C.L., Johnson, C.A., Critser, J.K., 2004. The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation. Hum. Reprod. 19, 1148–1154.
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
35
O’Neil, L., Paynter, S.J., Fuller, B.J., Shaw, R.W., Devries, A.L., 1998. Vitrification of mature mouse oocytes in a 6 M Me2 SO solution supplemented with antifreeze glycoproteins: the effect of temperature. Cryobiology 37, 59–66. Orief, Y., Schultze-Mosgau, A., Dafopoulos, K., Al-Hasani, S., 2005. Vitrification: will it replace the conventional gamete cryopreservation techniques? Middle East Fertil. Soc. J. 10, 171–184. Park, K.E., Kwon, I.K., Han, M.S., Niwa, K., 2005. Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes. J. Reprod. Dev. 51, 151–160. Rall, W.F., Reid, D.S., Polge, C., 1984. Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physiochemincal methods. Cryobiology 21, 106–121. Rojas, C., Palomo, M.J., Albarracın, J.L., Mogas, T., 2004. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 49, 211–220. Saito, N., Imai, K., Tomizawa, M., 1994. Effect of sugars addition on the survival of vitrified bovine blastocysts produced in vitro. Theriogenology 41, 1053–1060. Schroeder, A.C., Champlin, A.K., Mobraaten, L.E., Eppig, J.J., 1990. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fertil. 89, 43–50. Suzuki, T., Boediono, A., Takagi, M., Saha, S., Sumantri, C., 1996. Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro. Cryobiology 33, 515–524. Tada, N., Sato, M., Amann, E., Ogawa, S., 1993. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification—beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriogenology 40, 333–344. Tarin, J.J., Trounson, A.O., 1993. Effect of stimulation or inhibition of lipid peroxidation on freezing-thawing of mouse embryos. Biol. Reprod. 49, 1362–1368. Toth, T.L., Baka, S.G., Veeck, L.L., 1994. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil. Steril. 61, 891–894. Tucker, Wright, G., Morton, P.C., Massey, J.B., 1998. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil. Steril. 70, 578–579. Vincent, C., Turner, K., Pickering, S.J., Johnson, M.H., 1991. Zona pellucida modifications in the mouse in the absence of oocyte activation. Mol. Reprod. Dev. 28, 394–404. Wani, N.A., Maurya, S.N., Misra, A.K., Saxena, V.B., Lakhchaura, B.D., 2004. Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis). Theriogenology 61, 831–842. Wang, Y.R., Zhao, H., Sheng, X.H., Gambino, P.E., Costello, B., Bojanowski, K., 2002. Protective effect of fructus lycii polysaccharides against time and hyperthermia-induced damage in cultured seminiferous epithelium. J. Ethnopharmacol. 82, 169–175. Wray, W.T., Boulikas, T., Wray, V.P., Hancock, R., 1981. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–203. Zhao, R., Li, Q.W., Xiao, B., 2005. Effect of Lycium barbarum polysaccharide on the improvement of insulin resistance in NIDDM rats. Yakugaku Zasshi 125, 981–988.
Effect of sugars on maturation rate of vitrified-thawed immature porcine oocytes Jing Huang a , Qingwang Li a,b,∗ , Rui Zhao b , Wenye Li a , Zengsheng Han b , Xiaoyu Chen c , Bo Xiao a,d , Shuyun Wu a , Zhongliang Jiang a , Jianhong Hu a , Lei Liu b a
b
College of Animal Science, Northwest Agriculture and Forestry University, Yangling, Shannxi Province 712100, China Department of Biological Engineering, College of Enviroment and Chemical Engineering, Yanshan University, Qinhuangdao, Hebei Province 066004, China c Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province 310021, China d College of Life Sciences, LuDong University, Yantai, Shandong Province 264025, China Received 3 October 2006; received in revised form 21 December 2006; accepted 23 March 2007 Available online 30 March 2007
Abstract This study examined the effects of monosaccharide (glucose), disaccharide (sucrose) and polysaccharides (Ficoll and Lycium barbarum polysaccharide (LBP)) at different concentrations, using ethylene glycol (EG) as membrane-permeating cryoprotectant, on in vitro maturation of vitrified-thawed immature (GV) porcine oocytes. A total of 1145 oocytes were obtained by follicle aspiration from 496 ovaries of pigs slaughtered at a local abattoir and vitrified using a five-step method. After thawing and removal of cryoprotectant, oocytes were cultured for 44 h at 39 ◦ C in a humidified atmosphere of 5% CO2 in air. Oocytes were stained with DAPI and nuclear maturation was examined. The highest maturation rates were obtained in 1.5 M glucose (8.62%), 0.75 M sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.10 g/ml LBP (20.69%), respectively. The maturation rate using 0.75 M sucrose or 0.10 g/ml LBP was significantly higher compared to 1.5 M glucose (P < 0.05), but there was no significant difference from using 3.0 g/ml Ficoll (P > 0.05). The percentage of oocytes reaching metaphase II (MII) stage in the cryopreserved groups was significantly lower than control (P < 0.05). These results suggest that LBP is an effective non-permeating membrane cryoprotectant and 0.75 M sucrose or 0.10 g/ml LBP can be used as the vitrification solution for immature porcine oocytes. © 2007 Elsevier B.V. All rights reserved. Keywords: Porcine; Oocyte; Vitrification; Maturation ∗ Corresponding author. Present address: College of Animal Science, Northwest Agriculture and Forestry University, Yangling, Shannxi Province 712100, China. Tel.: +86 335 8074662; fax: +86 335 8074662. E-mail address: [email protected] (Q. Li).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.03.023
26
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
1. Introduction The rapid development of reproductive technologies, such as in vitro maturation, in vitro fertilization and in vitro embryo production has lead to increased demand for mammalian oocytes. Therefore, it would be a significant advance for basic research and commercial applications (Schroeder et al., 1990; Candy et al., 1994), if immature oocytes could be cryopreserved before maturation and use. Cryopreservation of immature oocytes has been reported with varying degrees of success in several mammalian species, such as mice (Eroglu et al., 1998; Moffa et al., 2002), cattle (Suzuki et al., 1996), buffalo (Wani et al., 2004) and humans (Tucker et al., 1998; Isachenko et al., 2006). However, immature porcine oocytes remain one of the most difficult to successfully cryopreserve due to their sensitivity to cooling and freezing. Many studies have showed that the in vitro maturation rate after vitrification is extremely low (Park et al., 2005; Rojas et al., 2004; Hara et al., 2005). Little success has been achieved with cryopreserving immature porcine oocytes. Isachenko et al. (1998) obtained 22% metaphase II (MII) stage oocytes after vitrification of immature (GV) oocytes by pretreating with cytochalasin B (CB). Fujihira et al. (2004) improved the maturation rate to 37.1% after vitrification of GV oocytes using a cryotop sheet. There are many studies involving cryoprotectants, but the role of sugars in the vitrification solution for immature (GV) porcine oocytes cryopreservation has not been evaluated. The protective action of sugars is very complex, attributable to a number of their special properties (Fabbri et al., 2000). Due to their large molecular size, sugars could cause an osmotic gradient across the cell membrane, which enhance dehydration of the cell before freezing of extracellular water. In addition, it is suggested that sugars are capable of preserving the structural and functional integrity of cell membranes at low water activities (Hotamisligil et al., 1996). As early as 1993, mouse embryos were vitrified with 2.75 M dimethyl sulfoxide and 2.75 M propylene glycol supplemented with 1.0 M sucrose after a 0.5-min exposure, which exhibited a significantly higher in vitro survival rate (82%) than the solution without 1.0 M sucrose (44%) (Tada et al., 1993). Furthermore, it has been reported that vitrification solution with added sugars could significantly improve the survival rate of the vitrified bovine blastocysts (Saito et al., 1994). Toth et al. (1994) also showed that the maturation rate of frozen human immature oocytes was improved when sugars were added. In a study of common carp embryos, it was clearly suggested that sugars lowered the toxicity of permeating cryoprotectants (Ahammad et al., 2002). In earlier studies, many commercial sugars were used widely as cryoprotectants, while over the last few decades, the biological activities of plant polysaccharides have attracted increased attention in biochemistry and medicine. However, to date, there is no report on the effects of plant polysaccharides as a cryoprotectant on the vitrification of immature porcine oocytes. The purpose of this study was to investigate the effects of monosaccharide (glucose), disaccharide (sucrose) and polysaccharides (Ficoll and LBP) at different concentrations on the post-thaw maturation rates of porcine oocytes and evaluate sugars as potential cryoprotectants for vitrification of porcine immature oocytes. To the best of our knowledge, this is the first report on the effect of Lycium barbarum polysaccharide on the vitrification of porcine immature oocytes. 2. Materials and methods Unless otherwise stated, all the chemicals used in this study were purchased from Sigma (St. Louis, MO, USA).
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
27
2.1. Preparation of L. barbarum polysaccharide Dried L. barbarum fruits were ground to a powder and the powder refluxed twice with petroleum ether (each time for 1 h) to remove lipids (Tianjin, China), then refluxed again with 80% ethanol (Tianjin, China) twice (each time for 2 h) to remove monosaccharides and oligosaccharides. The residue was extracted three times in hot water (90 ◦ C) and the combined filtrate concentrated through decompression using a rotary evaporator (Shanghai, China). A small amount of activated carbon (Qingdao, China) was added to remove color. After filtering, 95% ethanol was added to the filtrate and left overnight. The precipitate was washed sequentially using 95% ethanol, 100% ethanol, acetone and ether (Tianjin, China). Finally, the precipitate was collected and vacuum-dried to give the polysaccharide. 2.2. Ovaries selection and oocytes collection Ovaries were obtained from prepubertal pigs within 5 min after slaughtered at a local abattoir. The selected ovaries were in the follicular phase with no apparent corpus lutea and were transported to the laboratory in D-PBS containing 150 IU/ml penicillin and 100 g/ml streptomycin at 35 ◦ C within 2 h. The cumulus–oocyte complexes (COCs) were collected from non-atretic follicles (2–6 mm in diameter) by aspiration with an 18G needle attached to a 10-ml sterile syringe. Only oocytes with compact cumulus cells showing a homogeneous cytoplasm were selected and rinsed three times in TL–HEPES–PVA medium, as described previously (Funahashi et al., 1997). 2.3. Vitrification and thawing As shown in Table 1, the vitrification solutions were prepared using a holding medium (TCM199–Hepes supplemented with 20% fetal calf serum (FCS, Hyclone)). As ethylene glycol (EG) has low toxicity and good permeating capability, it was used as the cryoprotectant in this experiment. All manipulations were performed on a 41 ◦ C hot-plate in a room at 25–27 ◦ C to maintain the media at 38.5–39 ◦ C. Oocytes were first equilibrated in the holding medium for 5 min and then treated with 7.5 g/ml cytochalasin B for 25 min at 38.5 ◦ C. Oocytes were exposed to 5, 10, 20, 30 and 40% ethylene glycol, which corresponded to a 12.5, 25, 50, 75 and 100% sugar concentration of each treatment group for 3, 3, 2, 1 and 0.5 min, respectively (Table 1). Each group of 5–8 oocytes was loaded into 0.25 ml straws containing a 0.5-cm column of vitrification solution (40% EG), which were heat-sealed and immediately dipped in liquid nitrogen. After being stored in liquid nitrogen for at least 1 week, the straw was thawed in a water bath at 37 ◦ C for 5 s. The COCs were immediately expelled into a Petri dish with 2 ml of 0.75 M sucrose solution for 5 min. The oocytes were then transferred to the decreasing concentrations of 0.5 and 0.25 M sucrose Table 1 The component of vitrification solution and exposed time Steps
First step
Second step
Third step
Fourth step
Fifth step
Vitrification solution
5% EG (v/v) + 12.5% × A (M)
40% EG (v/v) + 100% × A (M)
3
20% EG (v/v) + 50% × A (M) 2
30% EG (v/v) + 75% × A (M)
Treat time (min)
10% EG (v/v) + 25% × A (M) 3
1
0.5
(EG) ethylene glycol and (A) the different sugar concentrations of treatment groups.
28
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
at 2-min intervals. The oocytes were subsequently washed three times in maturation medium, which was modified TCM-199 (TCM199; Gibco, Grand Island, NY, USA; Cat. No. 31100-035) supplemented with 0.1% PVA (Cat. No. P8136), 3.05 mM glucose (Ameresco, Cat. No. 0188), 0.91 mM sodium pyruvate (Cat. No. P5280), 0.57 mM/ml l-cysteine (Cat. No. C7352), 10 IU/ml PMSG (Cat. No. G4877), 10 IU/ml hCG (Cat. No. C1063), 10 ng/ml EGF (Promega, Cat. No. G5021), 50 IU/ml penicillin G and 50 IU/ml streptomycin sulfate. The morphological appearance of the oocytes was evaluated after thawing under an inverted microscope. The oocytes with spherical and symmetrical shape and no signs of lyses/degeneration were considered normal, whereas oocytes with ruptured zona pellucida, fragmented cytoplasm or degenerative signs were classified as abnormal. 2.4. In vitro maturation of the vitrified-thawed oocytes The washed COCs were kept in the oocyte maturation medium for 20 min in a CO2 incubator and then transferred to 500 l of the maturation medium in a four-well culture plate. The COCs were cultured for 44 h at 39 ◦ C in a humidified atmosphere of 5% CO2 in air. Fresh oocytes were also cultured in the same manner as control. 2.5. Assessment of nuclear maturation After a 44-h incubation in the maturation medium, oocytes with granulated ooplasm, broken membrane and zona pellucida were recorded as degenerated. Cumulus cells were removed by mixing oocytes with 0.1% hyaluronidase in phosphate-buffered saline (PBS). The nuclear status of the denuded oocytes was determined by DAPI (Cat. No. D9542) staining. The oocytes were fixed in PBS with 2% (w/v) formaldehyde, washed with PBS, stained with 2.5% (w/v) DAPI and mounted on slides (Mori et al., 1988). The nuclear state of the stained oocytes was assessed under a fluorescence microscope (TS100, Nikon, Japan) at 450 nm. Oocytes with diffused or slightly condensed chromatin were classified as being in the GV stage. Oocytes that possessed clumped or strongly condensed chromatin forming an irregular network of individual bivalents (prometaphase) or a metaphase plate but no polar body were classified as being in the MI stage. Oocytes with either a polar body or two bright chromatin spots were classified as being in the MII stage (Fig. 1). 2.6. Statistical analysis Each experiment was repeated four times. All data of each stage of meiosis were subjected to one-way ANOVA using SPASS (version 11.0 for Windows). Values were compared by a Duncan test for multiple comparisons. A probability of P < 0.05 was considered to be statistically significant. 3. Results Our experimental results showed that oocytes displayed severe dehydration when incubated with vitrification solution but that most of them could revert to normal when removed from the cryprotectants. A total of 14 zona pellucide breakages were noticed, 6 in glucose, 3 in sucrose, 3 in Ficoll and 2 in LBP. The percentage of oocytes reaching metaphase II stage was lower (P < 0.05) in the cryopreserved groups compared to control and the expansion effect of cummlus cell in the
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
29
Fig. 1. Porcine oocytes stained with DAPI and visualized under UV light. Germinal vesicle (A), metaphase I (B) and metaphase II (C) (×400).
30
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
Table 2 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of glucose Glucose concentrations (M)
Total oocytesa
GVb (%)
MIc (%)
MIId (%)
Dege (%)
2.0 1.5 1.0 0.5 Control
63 58 53 62 59
43a (68.25) 37a (63.79) 36a (67.92) 44a (70.97) 3b (5.08)
10a (15.87) 10a (17.24) 9a (16.98) 5b (8.06) 5b (8.47)
3a (4.76) 5a (8.62) 3a (5.67) 3a (4.84) 47b (79.66)
7ab (11.11) 6ab (10.34) 5ab (9.43) 10b (16.13) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times. b GV: germinal vesicle. c MI: metaphase I. d MII: metaphase II. e Deg: degenerate.
control group was better than that in the cryopreserved groups. In addition, the maturation rate of oocytes was low in vitrification solutions supplemented with low sugar concentrations. After culture, the percentage in GV stage was not significantly different in the groups with different concentrations of glucose or Ficoll. However, in the sucrose and LBP groups, significantly more GV stage ooctyes were found in the low compared to the high concentration groups. Regarding the effect of glucose on the maturation rates of vitrified-thawed immature porcine oocytes, as shown in Table 2, the highest rate was obtained in 1.5 M glucose (8.62%), and it was not significantly different from oocytes vitrified in any other concentration (P > 0.05). The percentage of oocytes reaching metaphase I stage in 2.0 M (15.87%), 1.5 M (17.24%) or 1.0 M (16.98%) glucose was higher than that in 0.5 M (8.06%) glucose and control (8.47%) (P < 0.05). With respect to the effect of sucrose on maturation rates of vitrified-thawed immature porcine oocytes, the results in Table 3 show that the highest maturation rate was obtained in 0.75 M sucrose (20.0%, P < 0.05), but it was not significantly different from those vitrified in 1.0 M (17.65%) and 0.5 M (18.97%) sucrose (P > 0.05). The percentage of oocytes reaching metaphase I stage was not significantly different in the groups vitrified using 1.0 M (17.65%), 0.75 M (16.67%) or 0.5 M (12.07%) sucrose (P > 0.05). As shown in Table 4, the maturation rate of oocytes in the Ficoll group was similar to those in the glucose and sucrose groups with increasing concentrations. The highest maturation rate was observed in the group vitrified in 3.0 g/ml Ficoll (13.79%), but it was not significantly different from the other concentrations of Ficoll (P > 0.05).
Table 3 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of sucrose Sucrose concentrations (M)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
1.0 0.75 0.5 0.25 Control
51 60 58 60 59
27a (52.94) 32ab (53.33) 35b (60.34) 43c (71.67) 3d (5.08)
9a (17.65) 10ab (16.67) 7abc (12.07) 3c (5.0) 5bc (8.47)
9ab (17.65) 12b (20.0) 11ab (18.97) 5a (8.33) 47c (79.66)
6a (11.76) 6a (10.0) 5a (8.62) 9a (15.0) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times.
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
31
Table 4 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of ficoll Ficoll concentrations (g/ml)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
4.0 3.0 2.0 1.0 Control
60 58 50 61 59
40a (66.66) 37a (63.79) 35a (70.0) 39a (63.94) 3b (5.08)
8a (13.33) 7a (12.07) 4a (8.0) 7a (11.48) 5a (8.47)
7a (11.67) 8a (13.79) 3a (6.0) 4a (6.56) 47b (79.66)
5a (8.33) 6a (10.34) 8ab (16.0) 11b (18.03) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times. Table 5 Maturation rates of vitrified-thawed immature porcine oocytes in different concentrations of L. barbarum polysaccharide L. barbarum polysaccharide concentrations (g/ml)
Total oocytesa
GV (%)
MI (%)
MII (%)
Deg (%)
0.10 0.075 0.05 0.025 Control
58 49 51 57 59
31a (53.45) 27a (55.10) 35ab (68.63) 39b (68.42) 3c (5.08)
10a (15.15) 8a (16.33) 5a (9.80) 7a (12.28) 5a (8.47)
12a (20.69) 9ab (18.37) 6b (11.76) 4b (7.02) 47c (79.66)
5a (8.62) 5a (10.20) 5a (9.80) 7a (12.28) 4a (6.78)
Within columns, values with different letters are significantly different (P < 0.05). a The experiment was replicated for four times.
Regarding the effect of LBP on maturation rates of vitrified-thawed immature porcine oocytes, the results in Table 5 show that the highest maturation rate was obtained in 0.10 g/ml LBP (20.69%), which was significantly higher than in 0.05 g/ml (11.76%, P < 0.05) and 0.025 g/ml (7.02%, P < 0.05), but it was not significantly different from those vitrified in 0.075 g/ml LBP (18.37%, P > 0.05). Furthermore, the expansion effect of cumulus cells in 0.10 and 0.075 g/ml LBP was superior compared to other cryopreserved groups. To assess the efficacy of sugars, four types were compared as to their effect on post-thaw oocyte maturation rates. The results are summarized in Fig. 2 and show that the highest maturation rate of vitrified-thawed porcine oocytes was obtained sequentially in 1.5 M glucose (8.62%), 0.75 M
Fig. 2. Maturation rates of vitrified-thawed immature porcine oocytes in vitrification solution adding 1.5 M glucose, 0.75 M sucrose, 3.0 g/ml Ficoll and 0.10 g/ml LBP. Values with different letters are significantly different (P < 0.05).
32
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.10 g/ml LBP (20.69%). The maturation rate of vitrified-thawed porcine oocytes using 1.5 M glucose was significantly lower than 0.75 M sucrose or 0.10 g/ml LBP (P < 0.05), but it was not significantly different from that in the 3.0 g/ml Ficoll added group (P > 0.05). 4. Discussion Cryoprotectants are divided into two types: membrane-permeating (e.g. ethylene glycol, dimethyl sulphoxide) and membrane non-permeating (e.g. sucrose, Ficoll). The membranepermeating cryoprotectants decrease the freezing point of the solution and prevent oocyte damage from high electrolyte concentrations (Rall et al., 1984). The membrane non-permeating cryoprotectants exerted their beneficial effects by cellular dehydration, caused by changing osmotic pressure. At present, the combined use of both cryoprotectants is standard. The present study attempted to evaluate the effect of varying levels of glucose, sucrose, Ficoll and LBP on the maturation rate of vitrified immature porcine oocytes. The experimental results showed that the best maturation rate was obtained by adding 0.10 g/ml LBP (20.69%), which is clearly higher than that reported by Rojas et al. (2004) in immature porcine oocytes (6.1%) vitrified in 40% EG + 0.5 M sucrose in open pulled straws (OPS). It is also higher than that reported by Park et al. (2005) in immature porcine oocytes (15.0%) vitrified after centrifugation and partial delipation. Vincent et al. (1991) suggested that sudden addition and removal of sucrose could result in significant zona pellucida hardening in MII stage mouse oocytes. In our study, the higher maturation rates of vitrified-thawed immature porcine oocytes may be related to the manner of exposure from low to high sugar concentrations. However, the maturation rates in this study were lower than those reported by Fujihira et al. (2004) for MII porcine oocytes (37.1%) employing the cryotop sheet and leading to rapid cooling rate of minimum volume. The maturation rate of vitrified-thawed immature porcine oocytes is lower than in other species (Wani et al., 2004; Moffa et al., 2002), the main reason being that immature porcine oocytes are very sensitive to cooling. Cytoskeleton elements in GV stage oocytes were straight and rigid, but in the MII stage they were undulating and flexible (Allworth and Albertini, 1993). In addition, due to high lipid levels in porcine oocytes, the hardened lipids produced by vitrification might also cause deformation and disruption of the cytoskeleton, leading to permanent damage (Orief et al., 2005). Sugars, as non-permeating cryoprotectants, have been used in the vitrification of oocytes or embryos of many species. The protective action of sugar is very complex and they could preserve the structural and functional integrity of the membrane at low water activities (Hotamisligil et al., 1996). Adding sugars to the vitrification solution could enhance viscosity, whereby incubation of cells in this solution before vitrification helps withdraw more water from the cells and reduce exposure of cells to the toxic effects of the cryoprotectants (Orief et al., 2005). Some reports have shown that the effect of added sugar was better than no sugar in the cryopreservation of human immature oocytes (Toth et al., 1994) and bovine blastocysts (Saito et al., 1994). Previous studies have demonstrated that monosaccharide was a more effective osmotic buffer than disaccharide (McWilliams et al., 1996). However, in our study, the maturation rate (8.62%) of vitrified-thawed immature porcine oocytes in the added glucose group was significantly lower than in the sucrose group (20.0%). It is hypothesized that osmotic stress in the vitrification solution supplemented with glucose was higher than that with sucrose, which could damage oocytes or that the temperature of vitrification in the solution with added sucrose was higher than the same concentration of added glucose (Kuleshova et al., 1999). In the present study, an oocyte maturation rate of 20.0%
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
33
was obtained using a medium containing 40% EG and 0.75 M sucrose, which was the highest of all four added sucrose groups and higher than that reported by Rojas et al. (2004) using the same vitrification solution. Polysaccharides could influence the viscosity of the vitrification solution and, thereby reduce the toxicity of the cryoprotectant through lowered concentration and prevent embryos from cryoinjury by reducing mechanical stress which occurs during cryopreservation (Dumoulin et al., 1994). Our observations show that the effect of Ficoll on the maturation rate of the vitrified-thawed immature porcine oocytes was not as good as with LBPs. Some reports have indicated that cryopreservation could damage antioxidant enzymes that protected cells against lipid peroxidation and freeze–thaw stress could be modified by incubating the embryo in the presence of inhibitors of membrane lipid peroxidation (Tarin and Trounson, 1993). LBP, extracted from the traditional Chinese herb L. barbarum, was found to have anticancer, antioxidant, hypoglycemic and immunological activities (Liu et al., 1996; Li et al., 1991; Zhao et al., 2005). LBP also protected seminiferous epithelium from structural damage and apoptosis in a testicular tissue culture system (Wang et al., 2002). Our experimental results suggest that adding LBP to vitrification solution might reduce the production of reactive oxygen species (ROS) and improve the antioxidant enzyme potential of oocytes, thereby preventing plasma membranes from lipid peroxidation and stabilizing membrane structure and permeability. LBP is a large polysaccharide molecule binding a protein, identified by ultraviolet, infrared spectroscopy and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Laemmli, 1970) and stained with Ag (Wray et al., 1981). Hays et al. (1996) reported that antifreeze glycoproteins (AFGPs) found in the blood of polar fish could inhibit leakage of intracellular contents across membranes during thermotropic phase transitions. O’Neil et al. (1998) reported that antifreeze glycoproteins could also be bound to the surface of growing ice-crystals, inhibiting the addition of any further water molecules. In this study, it was concluded that LBP exerted a similar effect to antifreeze glycoprotein. LBP also possesses enormous potential for structural variability, which could form a viscous matrix for encapsulation of the oocytes and preventing ice-crystal damage during cooling and warming. In this study, the maturation rates of vitrified-thawed immature porcine oocytes were superior in every group with added 1.5 M glucose (8.62%), 0.75 M sucrose (20.0%), 3.0 g/ml Ficoll (13.79%) and 0.1 g/ml LBP (20.69%). In groups with added glucose, sucrose and Ficoll, the maturation rates of vitrified-thawed immature porcine oocytes were higher with increasing sugar concentrations; however, maturation rates decreased when sugar concentrations reached a certain level in the vitrification solution. An appropriate osmotic stress in the vitrification solution is an important factor for improving the maturation rate of vitrified-thawed immature oocytes, which could be regulated by sugars. Fabbri et al. (2001) demonstrated that increasing the sucrose concentration from 0.1 to 0.3 M in a freezing medium improved the survival rate of human oocytes after vitrification and prevented the formation of intracellular ice. On the other hand, Mullen et al. (2004) verified that increased sucrose concentration imposed a greater osmotic stress and increased the likelihood of damage to the spindle. A proper balance between oocyte dehydration and spindle damage is necessary, which are both caused by the osmotic stress of sugars. LBP was difficult to dissolve in the vitrification solution compared to other sugars, such as Ficoll; therefore, we believe that the highest maturation rate of vitrified-thawed immature porcine oocytes in the LBP group has not been reached. The maturation rate of vitrified porcine immature oocytes would be improved, if the concentration of LBP can be increased. In conclusion, our experimental results have demonstrated that porcine oocytes could be vitrified successfully by supplementation with 0.75 M sucrose or 0.1 g/ml LBP. Further research is needed, such as purification of LBP for higher solubility in the vitrification solution, to find a more effective non-permeating cryoprotectant.
34
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
Acknowledgement This research was supported by the Institutes of Science and Technology of Qin Huangdao City of Hebei Province of China (No. D08). References Ahammad, M.M., Bhattacharyya, D., Jana, B.B., 2002. The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures. Cryobiology 44, 114–121. Allworth, A.E., Albertini, D.F., 1993. Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton. Dev. Biol. 158, 101–112. Candy, C.J., Wood, M.J., Whittingham, D.G., Merriman, J.A., Choudhury, N., 1994. Cryopreservation of immature mouse oocytes. Hum. Reprod. 9, 1738–1742. Dumoulin, J.C., Bergers-Janssen, J.M., Pieters, M.H., Enginsu, M.E., Geraedts, J.P., Evers, J.L., 1994. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil. Steril. 62, 793–798. Eroglu, A., Toner, M., Leykin, L., Toth, T.L., 1998. Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes. J. Assist. Reprod. Genet. 15, 447–454. Fabbri, R., Porcu, E., Marsella, T., Primavera, M.R., Rocchetta, G., Ciotti, P.M., Magrini, O., Seracchioli, R., Venturoli, S., Flamigni, C., 2000. Technical aspects of oocyte cryopreservation. Mol. Cell. Endocrinol. 169, 39–42. Fabbri, R., Porcu, E., Marsella, T., Roccetta, G., Venturoli, S., Flamigni, C., 2001. Human oocytes cryopreservation: new perspectives regarding oocyte survival. Hum. Reprod. 16, 411–416. Fujihira, T., Kishida, R., Fukui, Y., 2004. Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants. Cryobiology 49, 286–290. Funahashi, H., Cantley, T.C., Day, B.N., 1997. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol. Reprod. 57, 49–53. Hara, K., Abe, Y., Kumada, N., Aono, N., Kobayashi, J., Matsumoto, H., Sasada, H., Sato, E., 2005. Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitriffication. Cryobiology 50, 216–222. Hays, L.M., Feeney, R.E., Crowe, L.M., Crowe, J.H., Oliver, A.E., 1996. Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions. Biophysics 93, 6835–6840. Hotamisligil, S., Toner, M., Powers, R.D., 1996. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol. Reprod. 55, 161–168. Isachenko, V., Montag, M., Isachenko, E., Dessole, S., Nawroth, F., van der Ven, H., 2006. Aseptic vitrification of human germinal vesicle oocytes using dimethyl sulfoxide as a cryoprotectant. Fertil. Steril. 85, 741–747. Isachenko, V., Soler, C., Isachenko, E., Perez-Sanchez, F., Grishchenko, V., 1998. Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant. Cryobiology 36, 250–253. Kuleshova, L.L., MacFarlane, D.R., Trounson, A.O., Shaw, J.M., 1999. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38, 119–130. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680–685. Li, W., Pai, S.Z., Ma, W., 1991. Kinetic observation on the change of SOD, LPO, Hb in blood of old people with the Barbary Wolfberry (Lycium barbarum). Chin. Tradit. Herbal Drugs 22, 251–268. Liu, J.L., Zhang, L.H., Qian, Y.K., 1996. Immune tumor-inhabition of Lycium barbarum polysaccharide on S180-bearing mice. Chin. J. Immunol. 12, 115–117. McWilliams, R.B., Gibbons, W.E., Leibo, S.P., 1996. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Cryobiology 54, 1059–1069. Moffa, F., Comoglio, F., Krey, L.C., Grifo, J.A., Revelli, A., Massorbrio, M., Zhang, J., 2002. Germinal vesicle transfer between fresh and cryopreserved immature mouse oocytes. Hum. Reprod. 17, 178–183. Mori, C., Hashimoto, H., Hoshino, K., 1988. Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice. Biol. Reprod. 39, 737–742. Mullen, S.F., Agca, Y., Broermann, D.C., Jenkins, C.L., Johnson, C.A., Critser, J.K., 2004. The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation. Hum. Reprod. 19, 1148–1154.
J. Huang et al. / Animal Reproduction Science 106 (2008) 25–35
35
O’Neil, L., Paynter, S.J., Fuller, B.J., Shaw, R.W., Devries, A.L., 1998. Vitrification of mature mouse oocytes in a 6 M Me2 SO solution supplemented with antifreeze glycoproteins: the effect of temperature. Cryobiology 37, 59–66. Orief, Y., Schultze-Mosgau, A., Dafopoulos, K., Al-Hasani, S., 2005. Vitrification: will it replace the conventional gamete cryopreservation techniques? Middle East Fertil. Soc. J. 10, 171–184. Park, K.E., Kwon, I.K., Han, M.S., Niwa, K., 2005. Effects of partial removal of cytoplasmic lipid on survival of vitrified germinal vesicle stage pig oocytes. J. Reprod. Dev. 51, 151–160. Rall, W.F., Reid, D.S., Polge, C., 1984. Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physiochemincal methods. Cryobiology 21, 106–121. Rojas, C., Palomo, M.J., Albarracın, J.L., Mogas, T., 2004. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 49, 211–220. Saito, N., Imai, K., Tomizawa, M., 1994. Effect of sugars addition on the survival of vitrified bovine blastocysts produced in vitro. Theriogenology 41, 1053–1060. Schroeder, A.C., Champlin, A.K., Mobraaten, L.E., Eppig, J.J., 1990. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fertil. 89, 43–50. Suzuki, T., Boediono, A., Takagi, M., Saha, S., Sumantri, C., 1996. Fertilization and development of frozen-thawed germinal vesicle bovine oocytes by a one-step dilution method in vitro. Cryobiology 33, 515–524. Tada, N., Sato, M., Amann, E., Ogawa, S., 1993. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification—beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriogenology 40, 333–344. Tarin, J.J., Trounson, A.O., 1993. Effect of stimulation or inhibition of lipid peroxidation on freezing-thawing of mouse embryos. Biol. Reprod. 49, 1362–1368. Toth, T.L., Baka, S.G., Veeck, L.L., 1994. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil. Steril. 61, 891–894. Tucker, Wright, G., Morton, P.C., Massey, J.B., 1998. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil. Steril. 70, 578–579. Vincent, C., Turner, K., Pickering, S.J., Johnson, M.H., 1991. Zona pellucida modifications in the mouse in the absence of oocyte activation. Mol. Reprod. Dev. 28, 394–404. Wani, N.A., Maurya, S.N., Misra, A.K., Saxena, V.B., Lakhchaura, B.D., 2004. Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis). Theriogenology 61, 831–842. Wang, Y.R., Zhao, H., Sheng, X.H., Gambino, P.E., Costello, B., Bojanowski, K., 2002. Protective effect of fructus lycii polysaccharides against time and hyperthermia-induced damage in cultured seminiferous epithelium. J. Ethnopharmacol. 82, 169–175. Wray, W.T., Boulikas, T., Wray, V.P., Hancock, R., 1981. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–203. Zhao, R., Li, Q.W., Xiao, B., 2005. Effect of Lycium barbarum polysaccharide on the improvement of insulin resistance in NIDDM rats. Yakugaku Zasshi 125, 981–988.