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Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods
Cryobiology 69 (2014) 496–499
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Brief Communication
Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods q Nucharin Sripunya a,b, Yuanyuan Liang a,b, Kanchana Panyawai a,b, Kanokwan Srirattana a,b, Apichart Ngernsoungnern a,c, Piyada Ngernsoungnern a,c, Mariena Ketudat-Cairns a,b, Rangsun Parnpai a,b,⇑ a b c
Embryo Technology and Stem Cell Research Center, Thailand School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand School of Anatomy, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 2 September 2014 Available online 16 September 2014 Keywords: Bovine Immature oocytes Cryotop Solid surface vitrification Vitrification
a b s t r a c t The present study was undertaken to compare the efficacies of Cryotop (CT), solid surface vitrification (SSV) methods and cytochalasin B (CB) treatment for the cryopreservation of immature bovine oocytes, in terms of survival, nuclear maturation, and in vitro development. Solution exposed oocytes were in vitro maturated and fertilized. No difference was found in the rates of survival, nuclear maturation and blastocyst among solution exposed groups and fresh control group, except blastocysts rates in oocytes exposed to CB, cryoprotectant (CPA) and fluorescein diacetate (FDA) group (CB–CPA–FDA) (23%) significantly lower than that of control group (32%). CB pretreated ((+)CB) or non-pretreated (( )CB) COCs were vitrified either by SSV or CT. Among four vitrified groups the nuclear maturation rates (CT( )CB: 58%, CT(+)CB: 57%, SSV( )CB: 60%, SSV(+)CB: 63%), cleavage (CT( )CB: 36%, CT(+)CB: 24%, SSV( )CB: 34%, SSV(+)CB: 26%) and blastocysts rates (CT( )CB: 6%, CT(+)CB: 7%, SSV( )CB: 4%, SSV(+)CB: 6%) did not differ, but the rates of the four vitrified groups were significantly lower than those of non-vitrified group (81%, 71% and 26%, respectively). We thus conclude that CT and SSV perform equally in vitrification of bovine immature oocytes, and CB did not increase the viability, nuclear maturation, or in vitro development of vitrified oocytes. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Cryopreservation of bovine oocytes from slaughtered animals is of great importance for research and animal production purposes. The cytoskeletal relaxant cytochalasin B (CB) has been reported to reduce the microtubules damage by enhancing their stabilization during the vitrification process [3,7], and may be used to reduce chilling injury to oocytes and embryos. In spite of these findings, however, contradictory effects of CB have been reported in many species using different treatments [4]. Cryotop (CT) and solid surface vitrification (SSV) methods have been reported to be highly efficient for cryopreservation of bovine oocytes [1,2] SSV is an inexpensive method that reaches high cooling rates by using a
combination of microdrops and improved heat exchange through direct contact with a dry metal surface cooled by liquid nitrogen (LN) [2]. CT is one of the most successful ultra-rapid vitrification techniques that has resulted in excellent survival and developmental rates with human and bovine oocytes by using a plastic holder and a film strip [1]. Therefore, the objectives of this work was (1) to evaluate the toxicity of cryoprotective agents (CPA) and CB on the nuclear maturation and developmental competence of immature bovine oocytes, (2) to evaluate the efficiency of CT and SSV devices in cryopreserving immature bovine oocytes (with or without CB pretreatment) on oocytes nuclear maturation competence and in vitro development of the fertilization derived embryos. Materials and methods
q Statement of funding: This work was supported by Suranaree University of Technology and by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. ⇑ Corresponding author at: School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Fax: +66 4422 3164. E-mail address: [email protected] (R. Parnpai).
http://dx.doi.org/10.1016/j.cryobiol.2014.09.001 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory within 4 h and were kept in 0.9% (w/v) NaCl at room temperature. Cumulus-oocyte complexes (COCs) were collected from follicles 2–6 mm in diameter and COCs with homogenous cytoplasm were partially denuded by 0.2% hyaluronidase to be surrounded by 2 layers of cumulus cells.
N. Sripunya et al. / Cryobiology 69 (2014) 496–499
Vitrification was performed using CT (Kitazato Supply Co., Tokyo, Japan) or SSV methods. In brief, COCs were washed three times in base medium [BM; TCM199 supplemented with 20% fetal bovine serum (FBS, Gibco)] and then placed in BM containing 10% dimethylsulfoxide (Me2SO) (v/v) and 10% ethylene glycol (EG) (v/v) for 1 min at 22–24 °C. After this period they were transferred to BM containing 20% Me2SO, 20% EG, and 0.5 mol/L sucrose for 30 s at 22–24 °C. For the CT method, 5 COCs in a volume <0.1 lL were loaded onto the surface of the film strip of each CT and the CT was directly immersed in LN, protected with a cap. For the SSV method, groups of 5 COCs in approximately 2 lL of vitrification solution were dropped onto the LN-chilled dry surface of an aluminum foil, using a glass capillary and the vitrified droplets were then immersed in LN. Vitrified oocytes were warmed by transferring the SSV droplets or CT directly into a warming solution (BM containing 3 mL 0.5 mol/L sucrose) at 38.5 °C. 5 min later, the oocytes were transferred into BM for another 5 min. Warmed COCs were then washed in BM 3 times before evaluation of oocyte viability. Oocyte viability was assessed by fluorescein diacetate (FDA) staining. Briefly, COCs were treated with 2.5 lg/mL FDA in phosphate-buffered saline (PBS) supplemented with BSA for 2 min at 38.5 °C in a dark room. After that, oocytes were washed 3 times in PBS supplemented with BSA and evaluated under a fluorescence microscope (IX71; Olympus, Tokyo, Japan), using a U-MWIB3 filter with excitation wavelength of 460–495 nm and emission wavelength of 510 nm. Oocytes expressing bright green fluorescence were considered to be alive (FDA-positive) and used for IVM. FDA-positive COCs (20 per group) were cultured in 100 lL droplets of IVM medium (TCM199 supplemented with 10% FBS, 0.02 AU/mL follicle-stimulating hormone (AntrinÒ R.10 Kawasaki Pharmaceutical, Kawasaki, Japan), 50 IU/mL hCG (Chorulon; Intervet, Boxmeer, Netherlands), 10 ng/mL epidermal growth factor, 50 lmol/L cysteamine, and 1 lg/mL estradiol-17b) covered with mineral oil in a humidified atmosphere of 5% CO2 at 38.5 °C for 24 h. After 24 h of IVM, the COCs were denuded of cumulus cells by gentle pipetting in 0.2% hyarulonidase. To evaluate the nuclear maturation status, samples of oocytes were mounted on glass slides, immersed in a mixture of ethanol and acetic acid (3:1, v:v) for 24 h, and stained with 1% (w/v) aceto-orcein for observation. Oocytes with a visible nuclear membrane were classified as germinal vesicle (GV) stage. Oocytes beyond the GV stage were considered to have undergone germinal vesicle breakdown (GVBD). The absence of a visible nuclear membrane and the presence of condensed chromatin were considered to indicate the metaphase I (MI) stage. Oocytes with a metaphase plate and one polar body (PB) were classified as metaphase II (MII) stage. In vitro fertilization (IVF) was performed at 24 h of IVM, and frozen spermatozoa were thawed for 30 s in a 38 °C water bath before injected to the bottom of 4 mL snap tubes containing 2 mL of Tyrode albumin lactate pyruvate (TALP) medium and allowed to swim for 30 min. The supernatant was then centrifuged at 500g for 5 min, and the pellet was washed twice with IVF medium (TALP supplemented with 1 mM caffeine, 20 m mol/L penicillamine, 10 m mol/L hypotaurine, and 20 m mol/L epinephrine) by centrifugation at 500g for 5 min. Fertilization was performed in 50 lL IVF-medium containing 10 mature oocytes and sperm at 3 106/mL concentration for 12 h co-incubation. After that, oocytes were washed and cultured with mSOF medium in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 2 days at 38.5 °C (20 zygotes per 100 lL microdrop). Thereafter, embryos at the 8-cell stage were selected and co-cultured with bovine oviductal epithelium cells in a humidified atmosphere of 5% CO2 for 5 days at 38.5 °C until day 8. Half of the medium was renewed daily. Embryo development was recorded at the time of medium renewal. The cleavage rates were recorded on day 2.
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Experimental design Experiment 1: toxicity of FDA, CPA and CB To test the toxicity of FDA, CPA and CB, partially denuded COCs were separated into 5 groups: (1) COCs without any treatment (Fresh Control); (2) COCs stained by FDA (FDA exposed); (3) COCs exposed to 7.5 lg/mL CB for 15 min and stained by FDA (CB–FDA exposed); (4) COCs exposed to4 equilibration vitrification and warming solutions and stained by FDA (CPA–FDA exposed); (5) COCs exposed to CPA and pretreated with 7.5 lg/mL CB for 15 min and stained by FDA (CB–CPA–FDA exposed). After IVM, parts of the oocytes were fixed and stained to assess the nuclear maturation status. The rest of the IVM oocytes were fertilized in vitro, and their developmental abilities were compared to that of fresh control oocytes. Experiment 2: effects of CB and vitrifications method on nuclear maturation status of vitrified oocytes and embryo development of vitrified oocytes after IVM and IVF To evaluate the effects of CB and the efficiency of CT and SSV vitrification methods on the nuclear maturation status of vitrified oocytes subjected to IVM for 24 h, partially denuded COCs were separated into 5 groups: (1) COCs vitrified by CT method (CT( )CB); (2) COCs vitrified by CT method pretreated with 7.5 lg/mL CB for 15 min (CT(+)CB); (3) COCs vitrified by SSV method (SSV CB); (4) COCs vitrified by SSV method pretreated with 7.5 lg/mL CB for 15 min (SSV(+)CB). (5) COCs without vitrification (Control). After warming, surviving oocytes were subjected to IVM for 24 h, and their nuclear maturation status was evaluated after fixing and staining. The rest of IVM oocytes were subjected to IVF, and embryo development were compared to that of control oocytes. The experiments were replicated at least 5 times per treatment group. Data were analyzed by ANOVA using Statistical Analysis Systems software (SAS Inst. INC., Cary, NC, USA), and were considered to be statistically significant at P-values lower than 0.05. Results There was no significant difference in the oocytes survival rates among FDA exposed (100%), CB–FDA exposed (99%), CPA–FDA exposed (99%) and CB–CPA–FDA exposed (99%) groups. The status of nuclear maturation of solutions exposed oocytes is presented in Table 1. No significant difference was found in the rates of oocytes reaching MII stage among FDA exposed (81%), CB–FDA exposed (83%), CPA–FDA exposed (83%) and CB–CPA–FDA exposed (80%) groups, compared to Fresh control group (86%). The MII rates were unaffected by the solutions exposure. In IVF-derived embryonic development competence, the blastocyst rates of the Fresh control group (32%) was significantly higher than that of CB–CPA–FDA exposed group (23%), but blastocyst rates in CB–CPA–FDA exposed group did not significantly differ from those of FDA exposed (28%), CB–FDA exposed (30%) and CPA–FDA exposed (25%) groups (Table 2). In vitrified oocytes, no significant difference was found among CT( )CB, CT(+)CB, SSV( )CB and SSV(+)CB groups in the oocytes survival rates, which were all over 90%. The nuclear maturation rates to the M II stage were not statistically significant among CT( )CB (58%), CT(+)CB (57%), SSV( )CB (60%), and SSV(+)CB (63%) groups (Table 3). But the control group (81%) exhibited a significantly higher MII rate than those of four vitrified groups. The developmental ability of vitrified oocytes was shown in Table 4.
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N. Sripunya et al. / Cryobiology 69 (2014) 496–499
Table 1 In vitro nuclear maturation of immature bovine oocytes after FDA, CPA and CB exposure. Groups
No. of oocytes examined
Fresh Control FDA exposed CB–FDA exposed CPA–FDA exposed CB–CPA–FDA exposed
140 140 140 139 139
Nuclear status after IVM (% of live oocytes after IVM) GV
GVBD
MI
0 1 1 0 0
0 2 1 0 0
17 22 18 23 26
(0) (1) (1) (0) (0)
(0) (1) (1) (0) (0)
(12) (16) (13) (17) (19)
AI
TI
0 1 1 0 1
1 1 0 0 0
(0) (1) (1) (0) (1)
MII (1) (1) (0) (0) (0)
121 113 116 115 111
Abnormal (86) (81) (83) (83) (80)
1 0 3 1 1
(1) (0) (2) (1) (1)
No statistical difference was obtained (P > 0.05 ANOVA). FDA: fluorescein diacetate; CPA: cryoprotectant; CB: cytochalasin B; GV: germinal vesicle; GVBD: germinal vesicle breakdown; MI: metaphase I; AI: anaphase-I; TI: telophase-I; MII: metaphase II; abnormal: chromosome could not be detected by microscopy.
Table 2 In vitro embryo development of CPA and CB exposed bovine oocytes and quality of blastocyst following IVM–IVF. Groups
No. of oocytes IVF
Cleavage (%)
No. of oocytes developed to 8-C
Fresh Control FDA exposed CB–FDA exposed CPA–FDA exposed CB–CPA–FDA exposed
126 123 115 115 110
99 94 86 89 81
(79) (76) (75) (77) (74)
81 77 54 51 62
Mor
(64)a (63)a (47)c (44)c (56)b
52 47 36 34 26
BL
(41)a (38)a (31)b (30)b (24)c
40 34 34 29 25
(32)a (28)a,b (30)a,b (25)a,b (23)b
a,b,c
Means within columns with different superscripts differ (P < 0.05 ANOVA). FDA: fluorescein diacetate; CPA: cryoprotectant; CB: cytochalasin B; IVM: in vitro maturation; IVF: in vitro fertilization; 8-C: 8-cell stage; Mor: morula; BL: blastocyst.
Table 3 In vitro nuclear maturation of immature bovine oocytes after vitrification by CT and SSV methods. Groups
Fresh Control CT( )CB CT(+)CB SSV( )CB SSV(+)CB
No. of oocytes examined
140 151 142 139 132
Nuclear status after IVM (% of live oocytes after IVM) GV
GVBD
MI
1 4 4 5 5
2 2 0 0 1
22 54 54 49 42
(1) (3) (3) (4) (4)
(1) (1) (0) (0) (1)
(16)b (36)a (38)a (35)a (32)a
AI
TI
1 0 1 2 0
1 0 0 0 0
(1) (0) (1) (1) (0)
(1) (0) (0) (0) (0)
MII
Abnormal
113 (81)a 88 (58)b 81 (57)b 83 (60)b 83 (63)b
0 3 2 0 1
(0) (2) (1) (0) (1)
a,b
Means within columns with different superscripts differ (P < 0.05 ANOVA). CT: Cryotop; SSV: solid surface vitrification; CB: cytochalasin B; GV: germinal vesicle; GVBD: germinal vesicle breakdown; MI: metaphase I; AI: anaphase-I; TI: telophase-I; MII: metaphase II; Abnormal: chromosome could not be detected by microscopy.
Table 4 In vitro embryo development of mature bovine oocytes after vitrification by CT and SSV methods. Groups
No. of oocytes IVF
Cleavage (%)
No. of oocytes developed to 8-C
Fresh Control CT( )CB CT(+)CB SSV( )CB SSV(+)CB
122 155 149 151 146
87 56 35 52 38
(71)a (36)b (24)b (34)b (26)b
73 22 21 26 22
(60)a (14)b (14)b (17)b (15)b
Mor
BL
39 (32)a 13 (8)b 11 (7)b 10 (7)b 9 (6)b
32 (26)a 9 (6)b 10 (7)b 6 (4)b 9 (6)b
a,b
Means within columns with different superscripts differ (P < 0.05 ANOVA). CT: Cryotop; SSV: solid surface vitrification; CB: cytochalasin B; 8-C: 8-cell stage; Mor: morula; BL: blastocyst.
Although the cleavage rates did not differ among the four vitrified groups but all of them were significantly lower than that of the control group (Table 4). Similarly, the 8-C, morula, and blastocysts rates of the four vitrified groups were not different but significantly reduced compared with control group. Discussion In the present study, we demonstrated that FDA, CPA–FDA, CB–FDA or CB–CPA–FDA exposure did not affect the oocytes survival, and the viability of vitrified immature bovine oocytes was not improved by pretreatment with CB.
Cytochalasin B is a cell-permeable mycotoxin that inhibits microfilament polymerization and therefore organelle movements and nuclear extrusion in cells. Controversial results have been reported for the effects of CB on oocytes and embryo vitrification. CB pretreatment has been proven to increase the cryosurvival of oocytes and embryos in pigs [3]. In bovines, CB was found to be ineffective for the improvement of vitrification efficacy in matured bovine oocytes [6]. Similarly, reports in ovine [8] and buffalo immature oocytes [4] revealed that CB did not effectively improve survival, maturation or development rates following vitrification. In accordance with our results, which found that although there is no toxic effect on CB exposed oocytes, and CB did not improve
N. Sripunya et al. / Cryobiology 69 (2014) 496–499
the maturation rate and embryo development ability of vitrified immature bovine oocytes. This ineffectiveness of CB pretreatment on bovine oocytes may be due to some plausible explanation: (1) If addition and dilution of osmotically active permeable vitrification is done properly, the osmotic damage would be minimal. (2) If vitrification is done sufficiently fast, the cell cytoskeleton has no time to undergo any substantial perturbations. Ergo, if all the above is done, cytoskeleton would not be the main target, and thus, CB would not have any beneficial protective effect. High CPA concentrations are necessary to avoid ice-crystal formation in the vitrification protocol; however the toxicity of CPA is a limitation for the success of cryopreservation. In our present study, CPA–FDA exposed groups did not reduce the viability of immature oocytes and their ability to reach MII stage. The blastocyst development following IVF was impaired in the CB–CPA–FDA exposed group compared with the Fresh control group. In buffalo, although the viability of CPA exposed immature oocytes was impaired, the ability to reach the MII stage did not decrease compared with the control group [4]. GV oocytes are extremely sensitive to cryopreservation for reasons as yet unknown, and genetic materials remain confined and rigid in the nucleus. In our study, unlike the CB–CPA–FDA exposed group, CPA–FDA or CB–FDA alone did not have a toxic effect on the oocytes. We speculate that reduced development ability is due to the toxic effect of CB combined with CPA. Judging by nuclear maturation and embryo development competences, no significant difference was found between CT and SSV groups regardless the use of CB in our study. In buffalo, pretreatment with CB did not increase the viability, maturation or embryo development of vitrified immature oocytes, but CT showed superiority in terms of a higher survival and MII rate [4]. Our previous research examining different approaches to vitrification compared SSV with CT using bovine matured oocytes [9], and a similar vitrification solution to that used by Dinnyes et al. [2]. Although CT has been regarded as an efficient device, the high price and the limited number of oocytes that can be preserved on each CT sheet (5–6 oocytes/sheet), curtail its usefulness for the preservation of large amount of oocytes within a short time period. In regards to the effects of CT and SSV of this study, both methods served equally poorly in comparison to the fresh control. In our opinion, it indicates that the new methods of kinetic (hyper-fast) vitrification should be explored in the future as the current methods do not produce sufficiently fast cooling and warming rates. And as the result, inflict sublethal damage and hidden effects, thus, greatly impaired the quality of oocytes (only 1 of 14–1 of 25 oocytes developed to blastocysts in comparison to 1 of 4 for the fresh control). Oocytes vitrification has been reported to be accompanied by alterations in mitochondrial activity such as loss of activity or
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abnormal distribution [7]. The low frequency of blastocyst rates compared with the control reported here may be associated with the changes in lipid content and fatty acid composition in frozenthawed oocytes. Other possible factors may include loss or damage of cumulus cells, degradation of messenger RNA [10] and coolinginduced calcium efflux that causes DNA fragmentation [5]. In conclusion, our study revealed that CT and SSV perform equally in term of oocytes survival rate, nuclear maturation rates and subsequent development competence after IVF, and CB pretreatment did not effectively improve maturation and in vitro embryo developmental rates of vitrified immature bovine oocytes. Acknowledgments This work was supported by Suranaree University of Technology and by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. Yuanyuan Liang and Kanokwan Srirattana were supported by Suranaree University of Technology postgraduate research fellowship. References [1] R.C. Chian, M. Kuwayama, L. Tan, J. Tan, O. Kato, T. Nagai, High survival rate of bovine oocytes matured in vitro following vitrification, J. Reprod. Dev. 50 (2004) 685–696. [2] A. Dinnyés, Y. Dai, S. Jiang, X. Yang, High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer, Biol. Reprod. 63 (2000) 513–518. [3] V. Isachenko, C. Soler, E. Isachenko, F. Perez-Sanchez, V. Grishchenko, Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant, Cryobiology 36 (1998) 250–253. [4] Y. Liang, D. Rakwongrit, T. Phermthai, T. Somfai, T. Nagai, R. Parnpai, Cryopreservation of immature buffalo oocytes: effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods, Anim. Sci. J. 83 (2012) 630–638. [5] M. Mattioli, B. Barboni, L. Gioia, P. Loi, Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes, Mol. Reprod. Dev. 65 (2003) 289– 297. [6] A. Mezzalira, A.D. Vieira, D.P. Barbieri, M.F. Machado, A. Thaler Neto, M.L. Bernardi, C.A.M. Silva, M.I.B. Rubin, Vitrification of matured bovine oocytes treated with cytochalasin B, Theriogenology 57 (2002) 472 (Abstract). [7] G.J. Rho, S. Kim, J.G. Yoo, S. Balasubramanian, H.J. Lee, S.Y. Choe, Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes, Mol. Reprod. Dev. 63 (2002) 464–470. [8] M.A. Silvestre, J.Y. Aniz, I. Salvador, P. Santolaria, F. Lopez-Gatius, Vitrification of pre-pubertal ovine cumulus–oocyte complexes: effect of cytochalasin B pretreatment, Anim. Reprod. Sci. 93 (2006) 176–182. [9] N. Sripunya, T. Somfai, Y. Inaba, T. Nagai, K. Imai, R. Parnpai, A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes, J. Reprod. Dev. 56 (2010) 176–181. [10] S. Succu, D. Bebbere, L. Bogliolo, F. Ariu, S. Fois, G.G. Leoni, F. Berlinguer, S. Naitana, S. Ledda, Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance, Mol. Reprod. Dev. 75 (2008) 538–545.
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Brief Communication
Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods q Nucharin Sripunya a,b, Yuanyuan Liang a,b, Kanchana Panyawai a,b, Kanokwan Srirattana a,b, Apichart Ngernsoungnern a,c, Piyada Ngernsoungnern a,c, Mariena Ketudat-Cairns a,b, Rangsun Parnpai a,b,⇑ a b c
Embryo Technology and Stem Cell Research Center, Thailand School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand School of Anatomy, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 2 September 2014 Available online 16 September 2014 Keywords: Bovine Immature oocytes Cryotop Solid surface vitrification Vitrification
a b s t r a c t The present study was undertaken to compare the efficacies of Cryotop (CT), solid surface vitrification (SSV) methods and cytochalasin B (CB) treatment for the cryopreservation of immature bovine oocytes, in terms of survival, nuclear maturation, and in vitro development. Solution exposed oocytes were in vitro maturated and fertilized. No difference was found in the rates of survival, nuclear maturation and blastocyst among solution exposed groups and fresh control group, except blastocysts rates in oocytes exposed to CB, cryoprotectant (CPA) and fluorescein diacetate (FDA) group (CB–CPA–FDA) (23%) significantly lower than that of control group (32%). CB pretreated ((+)CB) or non-pretreated (( )CB) COCs were vitrified either by SSV or CT. Among four vitrified groups the nuclear maturation rates (CT( )CB: 58%, CT(+)CB: 57%, SSV( )CB: 60%, SSV(+)CB: 63%), cleavage (CT( )CB: 36%, CT(+)CB: 24%, SSV( )CB: 34%, SSV(+)CB: 26%) and blastocysts rates (CT( )CB: 6%, CT(+)CB: 7%, SSV( )CB: 4%, SSV(+)CB: 6%) did not differ, but the rates of the four vitrified groups were significantly lower than those of non-vitrified group (81%, 71% and 26%, respectively). We thus conclude that CT and SSV perform equally in vitrification of bovine immature oocytes, and CB did not increase the viability, nuclear maturation, or in vitro development of vitrified oocytes. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Cryopreservation of bovine oocytes from slaughtered animals is of great importance for research and animal production purposes. The cytoskeletal relaxant cytochalasin B (CB) has been reported to reduce the microtubules damage by enhancing their stabilization during the vitrification process [3,7], and may be used to reduce chilling injury to oocytes and embryos. In spite of these findings, however, contradictory effects of CB have been reported in many species using different treatments [4]. Cryotop (CT) and solid surface vitrification (SSV) methods have been reported to be highly efficient for cryopreservation of bovine oocytes [1,2] SSV is an inexpensive method that reaches high cooling rates by using a
combination of microdrops and improved heat exchange through direct contact with a dry metal surface cooled by liquid nitrogen (LN) [2]. CT is one of the most successful ultra-rapid vitrification techniques that has resulted in excellent survival and developmental rates with human and bovine oocytes by using a plastic holder and a film strip [1]. Therefore, the objectives of this work was (1) to evaluate the toxicity of cryoprotective agents (CPA) and CB on the nuclear maturation and developmental competence of immature bovine oocytes, (2) to evaluate the efficiency of CT and SSV devices in cryopreserving immature bovine oocytes (with or without CB pretreatment) on oocytes nuclear maturation competence and in vitro development of the fertilization derived embryos. Materials and methods
q Statement of funding: This work was supported by Suranaree University of Technology and by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. ⇑ Corresponding author at: School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. Fax: +66 4422 3164. E-mail address: [email protected] (R. Parnpai).
http://dx.doi.org/10.1016/j.cryobiol.2014.09.001 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
Bovine ovaries were obtained from a local slaughterhouse and transported to the laboratory within 4 h and were kept in 0.9% (w/v) NaCl at room temperature. Cumulus-oocyte complexes (COCs) were collected from follicles 2–6 mm in diameter and COCs with homogenous cytoplasm were partially denuded by 0.2% hyaluronidase to be surrounded by 2 layers of cumulus cells.
N. Sripunya et al. / Cryobiology 69 (2014) 496–499
Vitrification was performed using CT (Kitazato Supply Co., Tokyo, Japan) or SSV methods. In brief, COCs were washed three times in base medium [BM; TCM199 supplemented with 20% fetal bovine serum (FBS, Gibco)] and then placed in BM containing 10% dimethylsulfoxide (Me2SO) (v/v) and 10% ethylene glycol (EG) (v/v) for 1 min at 22–24 °C. After this period they were transferred to BM containing 20% Me2SO, 20% EG, and 0.5 mol/L sucrose for 30 s at 22–24 °C. For the CT method, 5 COCs in a volume <0.1 lL were loaded onto the surface of the film strip of each CT and the CT was directly immersed in LN, protected with a cap. For the SSV method, groups of 5 COCs in approximately 2 lL of vitrification solution were dropped onto the LN-chilled dry surface of an aluminum foil, using a glass capillary and the vitrified droplets were then immersed in LN. Vitrified oocytes were warmed by transferring the SSV droplets or CT directly into a warming solution (BM containing 3 mL 0.5 mol/L sucrose) at 38.5 °C. 5 min later, the oocytes were transferred into BM for another 5 min. Warmed COCs were then washed in BM 3 times before evaluation of oocyte viability. Oocyte viability was assessed by fluorescein diacetate (FDA) staining. Briefly, COCs were treated with 2.5 lg/mL FDA in phosphate-buffered saline (PBS) supplemented with BSA for 2 min at 38.5 °C in a dark room. After that, oocytes were washed 3 times in PBS supplemented with BSA and evaluated under a fluorescence microscope (IX71; Olympus, Tokyo, Japan), using a U-MWIB3 filter with excitation wavelength of 460–495 nm and emission wavelength of 510 nm. Oocytes expressing bright green fluorescence were considered to be alive (FDA-positive) and used for IVM. FDA-positive COCs (20 per group) were cultured in 100 lL droplets of IVM medium (TCM199 supplemented with 10% FBS, 0.02 AU/mL follicle-stimulating hormone (AntrinÒ R.10 Kawasaki Pharmaceutical, Kawasaki, Japan), 50 IU/mL hCG (Chorulon; Intervet, Boxmeer, Netherlands), 10 ng/mL epidermal growth factor, 50 lmol/L cysteamine, and 1 lg/mL estradiol-17b) covered with mineral oil in a humidified atmosphere of 5% CO2 at 38.5 °C for 24 h. After 24 h of IVM, the COCs were denuded of cumulus cells by gentle pipetting in 0.2% hyarulonidase. To evaluate the nuclear maturation status, samples of oocytes were mounted on glass slides, immersed in a mixture of ethanol and acetic acid (3:1, v:v) for 24 h, and stained with 1% (w/v) aceto-orcein for observation. Oocytes with a visible nuclear membrane were classified as germinal vesicle (GV) stage. Oocytes beyond the GV stage were considered to have undergone germinal vesicle breakdown (GVBD). The absence of a visible nuclear membrane and the presence of condensed chromatin were considered to indicate the metaphase I (MI) stage. Oocytes with a metaphase plate and one polar body (PB) were classified as metaphase II (MII) stage. In vitro fertilization (IVF) was performed at 24 h of IVM, and frozen spermatozoa were thawed for 30 s in a 38 °C water bath before injected to the bottom of 4 mL snap tubes containing 2 mL of Tyrode albumin lactate pyruvate (TALP) medium and allowed to swim for 30 min. The supernatant was then centrifuged at 500g for 5 min, and the pellet was washed twice with IVF medium (TALP supplemented with 1 mM caffeine, 20 m mol/L penicillamine, 10 m mol/L hypotaurine, and 20 m mol/L epinephrine) by centrifugation at 500g for 5 min. Fertilization was performed in 50 lL IVF-medium containing 10 mature oocytes and sperm at 3 106/mL concentration for 12 h co-incubation. After that, oocytes were washed and cultured with mSOF medium in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 2 days at 38.5 °C (20 zygotes per 100 lL microdrop). Thereafter, embryos at the 8-cell stage were selected and co-cultured with bovine oviductal epithelium cells in a humidified atmosphere of 5% CO2 for 5 days at 38.5 °C until day 8. Half of the medium was renewed daily. Embryo development was recorded at the time of medium renewal. The cleavage rates were recorded on day 2.
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Experimental design Experiment 1: toxicity of FDA, CPA and CB To test the toxicity of FDA, CPA and CB, partially denuded COCs were separated into 5 groups: (1) COCs without any treatment (Fresh Control); (2) COCs stained by FDA (FDA exposed); (3) COCs exposed to 7.5 lg/mL CB for 15 min and stained by FDA (CB–FDA exposed); (4) COCs exposed to4 equilibration vitrification and warming solutions and stained by FDA (CPA–FDA exposed); (5) COCs exposed to CPA and pretreated with 7.5 lg/mL CB for 15 min and stained by FDA (CB–CPA–FDA exposed). After IVM, parts of the oocytes were fixed and stained to assess the nuclear maturation status. The rest of the IVM oocytes were fertilized in vitro, and their developmental abilities were compared to that of fresh control oocytes. Experiment 2: effects of CB and vitrifications method on nuclear maturation status of vitrified oocytes and embryo development of vitrified oocytes after IVM and IVF To evaluate the effects of CB and the efficiency of CT and SSV vitrification methods on the nuclear maturation status of vitrified oocytes subjected to IVM for 24 h, partially denuded COCs were separated into 5 groups: (1) COCs vitrified by CT method (CT( )CB); (2) COCs vitrified by CT method pretreated with 7.5 lg/mL CB for 15 min (CT(+)CB); (3) COCs vitrified by SSV method (SSV CB); (4) COCs vitrified by SSV method pretreated with 7.5 lg/mL CB for 15 min (SSV(+)CB). (5) COCs without vitrification (Control). After warming, surviving oocytes were subjected to IVM for 24 h, and their nuclear maturation status was evaluated after fixing and staining. The rest of IVM oocytes were subjected to IVF, and embryo development were compared to that of control oocytes. The experiments were replicated at least 5 times per treatment group. Data were analyzed by ANOVA using Statistical Analysis Systems software (SAS Inst. INC., Cary, NC, USA), and were considered to be statistically significant at P-values lower than 0.05. Results There was no significant difference in the oocytes survival rates among FDA exposed (100%), CB–FDA exposed (99%), CPA–FDA exposed (99%) and CB–CPA–FDA exposed (99%) groups. The status of nuclear maturation of solutions exposed oocytes is presented in Table 1. No significant difference was found in the rates of oocytes reaching MII stage among FDA exposed (81%), CB–FDA exposed (83%), CPA–FDA exposed (83%) and CB–CPA–FDA exposed (80%) groups, compared to Fresh control group (86%). The MII rates were unaffected by the solutions exposure. In IVF-derived embryonic development competence, the blastocyst rates of the Fresh control group (32%) was significantly higher than that of CB–CPA–FDA exposed group (23%), but blastocyst rates in CB–CPA–FDA exposed group did not significantly differ from those of FDA exposed (28%), CB–FDA exposed (30%) and CPA–FDA exposed (25%) groups (Table 2). In vitrified oocytes, no significant difference was found among CT( )CB, CT(+)CB, SSV( )CB and SSV(+)CB groups in the oocytes survival rates, which were all over 90%. The nuclear maturation rates to the M II stage were not statistically significant among CT( )CB (58%), CT(+)CB (57%), SSV( )CB (60%), and SSV(+)CB (63%) groups (Table 3). But the control group (81%) exhibited a significantly higher MII rate than those of four vitrified groups. The developmental ability of vitrified oocytes was shown in Table 4.
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Table 1 In vitro nuclear maturation of immature bovine oocytes after FDA, CPA and CB exposure. Groups
No. of oocytes examined
Fresh Control FDA exposed CB–FDA exposed CPA–FDA exposed CB–CPA–FDA exposed
140 140 140 139 139
Nuclear status after IVM (% of live oocytes after IVM) GV
GVBD
MI
0 1 1 0 0
0 2 1 0 0
17 22 18 23 26
(0) (1) (1) (0) (0)
(0) (1) (1) (0) (0)
(12) (16) (13) (17) (19)
AI
TI
0 1 1 0 1
1 1 0 0 0
(0) (1) (1) (0) (1)
MII (1) (1) (0) (0) (0)
121 113 116 115 111
Abnormal (86) (81) (83) (83) (80)
1 0 3 1 1
(1) (0) (2) (1) (1)
No statistical difference was obtained (P > 0.05 ANOVA). FDA: fluorescein diacetate; CPA: cryoprotectant; CB: cytochalasin B; GV: germinal vesicle; GVBD: germinal vesicle breakdown; MI: metaphase I; AI: anaphase-I; TI: telophase-I; MII: metaphase II; abnormal: chromosome could not be detected by microscopy.
Table 2 In vitro embryo development of CPA and CB exposed bovine oocytes and quality of blastocyst following IVM–IVF. Groups
No. of oocytes IVF
Cleavage (%)
No. of oocytes developed to 8-C
Fresh Control FDA exposed CB–FDA exposed CPA–FDA exposed CB–CPA–FDA exposed
126 123 115 115 110
99 94 86 89 81
(79) (76) (75) (77) (74)
81 77 54 51 62
Mor
(64)a (63)a (47)c (44)c (56)b
52 47 36 34 26
BL
(41)a (38)a (31)b (30)b (24)c
40 34 34 29 25
(32)a (28)a,b (30)a,b (25)a,b (23)b
a,b,c
Means within columns with different superscripts differ (P < 0.05 ANOVA). FDA: fluorescein diacetate; CPA: cryoprotectant; CB: cytochalasin B; IVM: in vitro maturation; IVF: in vitro fertilization; 8-C: 8-cell stage; Mor: morula; BL: blastocyst.
Table 3 In vitro nuclear maturation of immature bovine oocytes after vitrification by CT and SSV methods. Groups
Fresh Control CT( )CB CT(+)CB SSV( )CB SSV(+)CB
No. of oocytes examined
140 151 142 139 132
Nuclear status after IVM (% of live oocytes after IVM) GV
GVBD
MI
1 4 4 5 5
2 2 0 0 1
22 54 54 49 42
(1) (3) (3) (4) (4)
(1) (1) (0) (0) (1)
(16)b (36)a (38)a (35)a (32)a
AI
TI
1 0 1 2 0
1 0 0 0 0
(1) (0) (1) (1) (0)
(1) (0) (0) (0) (0)
MII
Abnormal
113 (81)a 88 (58)b 81 (57)b 83 (60)b 83 (63)b
0 3 2 0 1
(0) (2) (1) (0) (1)
a,b
Means within columns with different superscripts differ (P < 0.05 ANOVA). CT: Cryotop; SSV: solid surface vitrification; CB: cytochalasin B; GV: germinal vesicle; GVBD: germinal vesicle breakdown; MI: metaphase I; AI: anaphase-I; TI: telophase-I; MII: metaphase II; Abnormal: chromosome could not be detected by microscopy.
Table 4 In vitro embryo development of mature bovine oocytes after vitrification by CT and SSV methods. Groups
No. of oocytes IVF
Cleavage (%)
No. of oocytes developed to 8-C
Fresh Control CT( )CB CT(+)CB SSV( )CB SSV(+)CB
122 155 149 151 146
87 56 35 52 38
(71)a (36)b (24)b (34)b (26)b
73 22 21 26 22
(60)a (14)b (14)b (17)b (15)b
Mor
BL
39 (32)a 13 (8)b 11 (7)b 10 (7)b 9 (6)b
32 (26)a 9 (6)b 10 (7)b 6 (4)b 9 (6)b
a,b
Means within columns with different superscripts differ (P < 0.05 ANOVA). CT: Cryotop; SSV: solid surface vitrification; CB: cytochalasin B; 8-C: 8-cell stage; Mor: morula; BL: blastocyst.
Although the cleavage rates did not differ among the four vitrified groups but all of them were significantly lower than that of the control group (Table 4). Similarly, the 8-C, morula, and blastocysts rates of the four vitrified groups were not different but significantly reduced compared with control group. Discussion In the present study, we demonstrated that FDA, CPA–FDA, CB–FDA or CB–CPA–FDA exposure did not affect the oocytes survival, and the viability of vitrified immature bovine oocytes was not improved by pretreatment with CB.
Cytochalasin B is a cell-permeable mycotoxin that inhibits microfilament polymerization and therefore organelle movements and nuclear extrusion in cells. Controversial results have been reported for the effects of CB on oocytes and embryo vitrification. CB pretreatment has been proven to increase the cryosurvival of oocytes and embryos in pigs [3]. In bovines, CB was found to be ineffective for the improvement of vitrification efficacy in matured bovine oocytes [6]. Similarly, reports in ovine [8] and buffalo immature oocytes [4] revealed that CB did not effectively improve survival, maturation or development rates following vitrification. In accordance with our results, which found that although there is no toxic effect on CB exposed oocytes, and CB did not improve
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the maturation rate and embryo development ability of vitrified immature bovine oocytes. This ineffectiveness of CB pretreatment on bovine oocytes may be due to some plausible explanation: (1) If addition and dilution of osmotically active permeable vitrification is done properly, the osmotic damage would be minimal. (2) If vitrification is done sufficiently fast, the cell cytoskeleton has no time to undergo any substantial perturbations. Ergo, if all the above is done, cytoskeleton would not be the main target, and thus, CB would not have any beneficial protective effect. High CPA concentrations are necessary to avoid ice-crystal formation in the vitrification protocol; however the toxicity of CPA is a limitation for the success of cryopreservation. In our present study, CPA–FDA exposed groups did not reduce the viability of immature oocytes and their ability to reach MII stage. The blastocyst development following IVF was impaired in the CB–CPA–FDA exposed group compared with the Fresh control group. In buffalo, although the viability of CPA exposed immature oocytes was impaired, the ability to reach the MII stage did not decrease compared with the control group [4]. GV oocytes are extremely sensitive to cryopreservation for reasons as yet unknown, and genetic materials remain confined and rigid in the nucleus. In our study, unlike the CB–CPA–FDA exposed group, CPA–FDA or CB–FDA alone did not have a toxic effect on the oocytes. We speculate that reduced development ability is due to the toxic effect of CB combined with CPA. Judging by nuclear maturation and embryo development competences, no significant difference was found between CT and SSV groups regardless the use of CB in our study. In buffalo, pretreatment with CB did not increase the viability, maturation or embryo development of vitrified immature oocytes, but CT showed superiority in terms of a higher survival and MII rate [4]. Our previous research examining different approaches to vitrification compared SSV with CT using bovine matured oocytes [9], and a similar vitrification solution to that used by Dinnyes et al. [2]. Although CT has been regarded as an efficient device, the high price and the limited number of oocytes that can be preserved on each CT sheet (5–6 oocytes/sheet), curtail its usefulness for the preservation of large amount of oocytes within a short time period. In regards to the effects of CT and SSV of this study, both methods served equally poorly in comparison to the fresh control. In our opinion, it indicates that the new methods of kinetic (hyper-fast) vitrification should be explored in the future as the current methods do not produce sufficiently fast cooling and warming rates. And as the result, inflict sublethal damage and hidden effects, thus, greatly impaired the quality of oocytes (only 1 of 14–1 of 25 oocytes developed to blastocysts in comparison to 1 of 4 for the fresh control). Oocytes vitrification has been reported to be accompanied by alterations in mitochondrial activity such as loss of activity or
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abnormal distribution [7]. The low frequency of blastocyst rates compared with the control reported here may be associated with the changes in lipid content and fatty acid composition in frozenthawed oocytes. Other possible factors may include loss or damage of cumulus cells, degradation of messenger RNA [10] and coolinginduced calcium efflux that causes DNA fragmentation [5]. In conclusion, our study revealed that CT and SSV perform equally in term of oocytes survival rate, nuclear maturation rates and subsequent development competence after IVF, and CB pretreatment did not effectively improve maturation and in vitro embryo developmental rates of vitrified immature bovine oocytes. Acknowledgments This work was supported by Suranaree University of Technology and by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission. Yuanyuan Liang and Kanokwan Srirattana were supported by Suranaree University of Technology postgraduate research fellowship. References [1] R.C. Chian, M. Kuwayama, L. Tan, J. Tan, O. Kato, T. Nagai, High survival rate of bovine oocytes matured in vitro following vitrification, J. Reprod. Dev. 50 (2004) 685–696. [2] A. Dinnyés, Y. Dai, S. Jiang, X. Yang, High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer, Biol. Reprod. 63 (2000) 513–518. [3] V. Isachenko, C. Soler, E. Isachenko, F. Perez-Sanchez, V. Grishchenko, Vitrification of immature porcine oocytes: effects of lipid droplets, temperature, cytoskeleton, and addition and removal of cryoprotectant, Cryobiology 36 (1998) 250–253. [4] Y. Liang, D. Rakwongrit, T. Phermthai, T. Somfai, T. Nagai, R. Parnpai, Cryopreservation of immature buffalo oocytes: effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods, Anim. Sci. J. 83 (2012) 630–638. [5] M. Mattioli, B. Barboni, L. Gioia, P. Loi, Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes, Mol. Reprod. Dev. 65 (2003) 289– 297. [6] A. Mezzalira, A.D. Vieira, D.P. Barbieri, M.F. Machado, A. Thaler Neto, M.L. Bernardi, C.A.M. Silva, M.I.B. Rubin, Vitrification of matured bovine oocytes treated with cytochalasin B, Theriogenology 57 (2002) 472 (Abstract). [7] G.J. Rho, S. Kim, J.G. Yoo, S. Balasubramanian, H.J. Lee, S.Y. Choe, Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes, Mol. Reprod. Dev. 63 (2002) 464–470. [8] M.A. Silvestre, J.Y. Aniz, I. Salvador, P. Santolaria, F. Lopez-Gatius, Vitrification of pre-pubertal ovine cumulus–oocyte complexes: effect of cytochalasin B pretreatment, Anim. Reprod. Sci. 93 (2006) 176–182. [9] N. Sripunya, T. Somfai, Y. Inaba, T. Nagai, K. Imai, R. Parnpai, A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes, J. Reprod. Dev. 56 (2010) 176–181. [10] S. Succu, D. Bebbere, L. Bogliolo, F. Ariu, S. Fois, G.G. Leoni, F. Berlinguer, S. Naitana, S. Ledda, Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance, Mol. Reprod. Dev. 75 (2008) 538–545.