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Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics
Cryobiology 65 (2012) 278–283
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Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics q V. Asgari a, S.M. Hosseini a, M. Forouzanfar b, M. Hajian a, M.H. Nasr-Esfahani a,c,⇑ a
Department of Reproduction and Development, Reproductive Biomedicine Centre, Royan Institute of Biotechnology, ACECR, Isfahan, Iran Department of Basic Science, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran c Department of Embryology, Reproductive Biomedicine Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran b
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Article history: Received 18 February 2012 Accepted 8 August 2012 Available online 21 August 2012 Keywords: Bovine Embryo Vitrification Embryonic genome activation Kinetics
a b s t r a c t In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8–16 cell stage): I and II – early (day 2) and late (day 3) 5–8 cell, III and IV – early (day 3) and late (day 4) 8–16 cell, and V and VI – early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5–8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8–16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Successful cryopreservation of embryos was first reported in 1971 in mice [36]. Increased possibility of embryo transfer technology in domestic animals and then human resulted in a parallel growing focus of interest to cryopreserve embryos in these species [27,37]. However, one dilemma that has remained to be addressed is the optimal embryonic stage (early stage vs. blastocyst) at which the embryos are preserved [22]. Theoretically, blastocyst stage embryos have overcome the embryonic block and a first selection has already been made. These embryos have greater nuclear cytoplasmic ratio which makes them more suitable for cryopreservation [20]. The embryo developmental block is a phenomenon that occurs in many species at different stages (for example third to fourth cell Abbreviations: EGA, embryonic genome activation; COC, cumulus oocyte complex; VS, vitrification solution; Me2SO, dimethyl sulfoxide; EG, ethylene glycol; RT, room temperature; ES, equilibration solution; TCN, total cell number; ICM, inner cell mass; TE, trophectoderm. q Statement of funding: This study was performed by the Grant of Royan Institute. There is no conflict of interest in this study. ⇑ Corresponding author at: Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran. Fax: + 98 311 2605525. E-mail addresses: [email protected], [email protected] (M.H. Nasr-Esfahani).
0011-2240/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2012.08.002
cycle in humans, second cell cycle in mice and fourth or between the fourth and fifth cell cycle transition in bovine) due to the inability to activate zygotic genes and continue cleavage [30]. This species-specific block moment is associated with the developmental stage when embryos have to rely on the mRNAs transcribed from its own genome to continue cleavage [21]. In practice, the rate of ‘‘take home baby rate’’ per vitrified– warmed embryo at blastocyst stage is higher than embryos which are vitrified at earlier stages of development [22]. However, considering the potential disadvantages of prolonged in vitro embryo culture, there is an indispensable focus on vitrification of early stages embryos [4,22,24]. Although vitrification of early stage has been a matter of intensive focus in recent years, the impact of embryonic block and developmental kinetics (timing of first and subsequent embryonic cleavage) has been less noticed. It is well established that early embryonic events are orchestrated through the post-transcriptional control of maternal mRNA that have been accumulated during oogenesis [31]. These maternal mRNA and protein serve the distinct purpose of reprogramming and facilitating the early mitotic divisions of the fertilized embryos until broad embryonic genome activation (EGA) that is initiated at a species-specific stage of embryo development [3,6], so that the precise control over the time of EGA is essential for normal embryogenesis. Importantly, a significant reduction in mRNA content of vitrified–warmed early embryos has been documented in
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different species [5,32]. Therefore, considering the critical importance of maternally reserved mRNA for early embryo development, it is vital to determine if vitrification of embryos before or after EGA may affect cryosurvival of embryos. This approach requires an animal model with late embryonic genome activation. It is also well established that the time of the first cleavage postinsemination has major, long-lasting effects on subsequent development of embryos [14]. The rate of blastocyst development and the cell number of the resulting embryos have been related to the time of the first cleavage [14,7]. The evidences from both human and non-human studies suggest that early-cleaved embryos produced blastocysts, are more likely to survive cryopreservation and establishment pregnancies following embryo transfer than blastocyst derived from late cleaved embryos [22,28,38]. However, it is not clearly comprehended that how the step-wise kinetics of embryo development during different stages of preimplantation may affect cryosurvival of the embryos. Such studies would lead, in practice, to design more efficient strategies for selection the optimal stage of embryos development for vitrification. Therefore, the objective of the present study was to investigate the effect of developmental stage and kinetics of embryo development on cryosurvival of in vitro produced bovine embryos. Materials and methods Unless otherwise stated, all chemicals and media were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Gibco (Grand Island, NY, USA), respectively. In vitro oocyte maturation The process of oocyte IVM was as described previously [23]. In brief, ovaries from adult cows obtained from a local slaughterhouse, placed in saline (35 °C) and transported to the laboratory
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within 3–5 h. Cumulus–oocyte complexes (COCs) were aspirated from antral follicles (2–8 mm in diameter) using 18-gauge needles attached to vacuum pump (80 mmHg). COCs with homogenous cytoplasm and more than three layers of cumulus cells were then matured in tissue culture medium 199 (TCM199) containing 2.5 mM Na-pyruvate, 100 IU/ml penicillin, 100 lg/ml streptomycin, 1 mg/ml estradiol-17b, 10 lg/ml FSH, 10 lg/ml LH, 100 ng/ml EGF, 0.1 mM cysteamine and 10% fetal calf serum (FCS) at 39 °C in a humidified atmosphere containing 5% CO2 (C200, Labotect, Germany). Sperm preparation and in vitro embryo development Frozen–thawed and washed sperm from a single Holstein sire of proven in vitro fertility were used for fertilization after capacitation by the swim-up procedure. Spermatozoa (1 106 sperms/ ml) and matured COCs (40–45 COCs/200 ll) were co-incubated in modified fert-TALP medium containing 0.01 mM heparin, 0.2 mM penicillamine, and 0.1 mM hypotaurine at 38.5 °C for 18– 24 h under 5% CO2 in humidified air. The presumptive zygotes were then vortexed for 90 s in Hepes-TALP to remove the cumulus cells. After that, they were washed once in H-TCM199 and twice in synthetic oviductal fluid (SOF) and then cultured as described above. Incubation was performed at 38.5 °C, under 5% CO2 and 5% O2 in humidified air [11]. Experimental design From day 3 onwards, cleaved embryos at different stages of development were sorted based on the kinetics of embryo development. Accordingly, early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. Early and late embryos at each stage were used for vitrification and control
Fig. 1. Diagrammatic representation of experimental design. Cleaved embryos at early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. Early and late embryos at each stage were used for vitrification and control experiments. E: early, L: late, UN: un-vitrified, V: vitrified. Bar represents 50 lm.
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experiments. After that, the vitrified embryos were immediately warmed and cultured along with their corresponding control groups until the blastocyst stage (Fig. 1). Vitrification of embryos The vitrification protocol used in this study was as described previously [10]. Briefly, embryos were pre-equilibrated in a solution of 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (Me2SO) in phosphate buffer saline free of calcium and magnesium (PBS ) supplemented with 20% FCS until the expanded blastocysts endured a period of shrinkage and returning (up to 8 min, at 38.5 °C). Equilibrated embryos were then exposed to vitrification solution consisting of 15% EG + 15% Me2SO + 0.5 M sucrose in holding medium at room temperature for 1 min, and then were loaded on the tips of the cryotops (Cryologic; CVM™, Fibreplug & Sleeve, Australia) with the minimum amount of vitrification solution and immediately plunged into the liquid nitrogen (LN2). For warming, embryos were removed from LN2 and quickly exposed into PBS + 20% FCS supplemented with 1 M sucrose for 1 min on a warm plate (38.5 °C). Then, embryos were transferred to PBS + 20% FCS containing 0.5 M sucrose for 3 min and finally washed thoroughly in PBS + 20% FCS before being cultured in SOF medium.
calculation of blastocyst formation rate is increased. Accordingly, although the potential of 5–8 cell stage embryos to survive vitrification and to further develop to the blastocyst stage was insignificantly lower than its corresponding control (un-vitrified 5–8 cell stage embryos), but it was significantly lower than vitrified, un-vitrified 8–16 cell and morula stage embryos (Fig. 2A), while these differences were not considerable for un-vitrified 5–8 cell stage embryos. Neither the vitrification process nor the stage of embryo development at the time of vitrification affected the hatching ability of the developed blastocysts (Fig. 2B). Moreover, differential analysis of ICM and TE did not reveal any significant difference between the vitrified and un-vitrified or the stage at which the embryos were frozen (Fig. 2C). Effect of developmental kinetics on cryosurvival Within each developmental stage, neither vitrification–warming nor the kinetics of embryo development affected their survival
Differential embryo staining for quality assessment In order to determine the total cell number (TCN) and the sole number of cells allocated in the sites of inner cell mass (ICM) and trophectoderm (TE), the hatched blastocysts in both groups were assigned to differential staining as described elsewhere [23]. In brief, hatched blastocysts were incubated in 500 ll of 1% Triton X-100 and 100 lg/ml propidium iodide (solution 1) for up to 30 s, depending on the size of the embryos, and then immediately transferred into a 500 ll solution of 100% ethanol plus 25 lg/ml Hoechst 33258 (solution 2). Care was taken to carry the minimum amount of solution 1 when the embryos were transferred into solution 2. The embryos were extensively washed in solution 2 to remove any trace amounts of solution 1. Samples were then stored in solution 2 at 4 °C overnight. Fixed and stained embryos were subsequently mounted onto a glass slide in one drop of glycerol, gently flattened with a cover slip and visualized for cell counting on a fluorescence microscope (excitation filter 460 nm for blue and 560 nm for red). TE cells were visualized as blue and ICM as pink to red. TCN was calculated by counting the numbers of both ICM and TE. Statistical analysis All of the experiments in the present study were repeated at least three times. Percentages data were modeled to the binomial model of parameters by ArcSin transformation. The transformed data were analyzed by one way ANOVA model of SPSS 17. Also, differences were compared by Tukey multiple comparison post hoc test. It is noteworthy to mention that all data were presented as means ± SEM and differences considered significant at P 6 0.05. Results Effect of developmental stage on cryosurvival Fig. 2 represents the effect of stage of embryo development on cryosurvival of the resultant embryos. As shown, irrespective of the vitrification procedure, developmental competence of embryos was increased as the developmental stage taken into account for
Fig. 2. Effect of developmental stage and vitrification on blastocyst formation, hatching rate and the number of inner cell mass (ICM) cells per total cells of bovine in vitro produced (IVP) embryos. V: vitrified, UV: un-vitrified. Different letters display significant differences at P 6 0.05.
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Fig. 3. Effect of developmental stage and vitrification on the kinetics of blastocyst formation of bovine IVP embryos (early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively). V: vitrified, UV: un-vitrified. Different letters display significant differences at P 6 0.05.
and subsequent potential to develop to the blastocyst stage (Fig. 3). Furthermore, the hatching ability (Fig. 4) and differential analysis of ICM and TE (Fig. 5) did not reveal any significant difference between vitrified and un-vitrified embryos. Moreover, differential allocations of ICM and TCN were not affected by developmental kinetics, irrespective of the day at which the blastocysts were formed. Discussion Literature study clearly revealed that pregnancy rate is substantially higher when blastocyst is vitrified–warmed and then transferred compared to transfer of vitrified–warmed early stage embryos [1,12,13,25,29,33]. There are several important reasons
Fig. 4. Effect of developmental stage and vitrification on the kinetics of hatching rate of bovine early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. V: vitrified, UV: un-vitrified.
for this difference, including higher cell number and increased nucleo-to-cytoplasmic ratio in blastocysts which guarantees optimum equilibrium with the cryoprotectants and allows an embryo recovery even if some cells are destroyed in response to cryodamages [22,16]. Another stark difference between blastocysts and early stage embryos is that the blastocysts have already overcome the species-specific genomic activation block and a first selection has already been made between developing and
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Fig. 5. Effect of developmental stage and vitrification on the number of inner cell mass (ICM) cells per total cell number (TCN) of resultant blastocyst (at day 8 and 9) of bovine early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. V: vitrified, UV: un-vitrified.
non-developing embryos [20]. Despite the latter facts, the link between developmental kinetics, time of embryonic genome activation and cryosurvival of early bovine embryos remains to be defined. The results of this study showed that developmental stage had a deterministic impact on developmental competency of embryos which have survived vitrification to produced blastocyst in vitro. Accordingly, considering the time of embryo genome activation which corresponded to around 16 cell stage in bovine, it was ob-
served that vitrification of embryos before EGA (at 5–8 cell stage) significantly reduced their developmental competence compared to those embryos that were vitrified–warmed at the later stages or beyond EGA (8–16 cell or the morula stage). However, it is important to note that un-vitrified embryos at 5–8 cell stage did not show a significant reduction in the developmental competence as compared with later stage embryos, whereas, interestingly the majority of embryos that were vitrified–warmed at 5–8 cell stage arrested before morula stage. Two main mechanisms may describe the aforementioned difference: (i) inability to overcome the chromatin repression and activate transcription of important developmental genes and/or (ii) to react to injuries caused by environment [2], such as vitrification–warming. Cryopreservation method can be influence the developmental competency of vitrified–warmed embryos at different developmental stage. In this regard, Mandelbaum et al. [19] showed that optimal survival rates are only achieved when sucrose is combined with propanediol (PrOH) and propanediol itself leads to reduced survival rates. On the other hand, Van der Elst et al. [34] demonstrated that vitrification of multicellular embryos could be improved with DMSO. However, the success rates reported with the PrOH protocol were considerably low. The reduction of the developmental competence of vitrified vs. non-vitrified 5–8 cell stage embryos in this study can be described from two different perspectives; cellular and molecular. From cellular point of view, it is well established that the embryonic divisions is concomitant with a reduction in cellular volume [15] and it provides a better equilibration of the embryos with cryoprotectants, and thereby better cryo-withstand. Furthermore, embryo progression results in a subsequent increase in nucleo-to-cytoplasmic ratio which is beneficial to cryopreservation [16]. From the molecular point of view, vitrification of embryos at earlier stages of development before EGA may deprive embryos of maternal reserve of mRNAs that are responsible for completion of embryo development before EGA initiation. It is interesting that mouse embryos vitrified at two cell stage (time of EGA in mice) have had better cryosurvival rate than those vitrified before EGA (at zygotic stage) [8]. However, to provide a better understanding of the impact of the mentioned mechanisms, comparative analysis of the transcriptomics, proteomics, and metabolomics of the early embryos before and after vitrification–warming are beneficial. Kinetics of early embryo development is another important embryo characteristic which allows prediction of the potential of embryos to develop towards blastocyst stage [17]. Accordingly, it has been demonstrated that the rate of blastocyst development and the cell number of the resulting embryos correlate with the time of the first cleavage [35,18]. It has been also shown that timing of the first cleavage post-insemination can influence the cryosurvival of bovine blastocysts following vitrification [9]. Moreover, it has been indicated that fast developing embryos (expanded blastocyst and early hatching blastocyst stage) had better resistance to freezing than delayed ones (early blastocyst stage) derived from the same group of embryos which were inseminated concomitantly [26]. However, it was not clear if kinetics of embryo development beyond two cells stage can also affect cryosurvival and subsequent development. The results of this study indicated that for each developmental stage, kinetics of embryo development had no predictive value for embryos further development. Accordingly, all the parameters analyzed in this study including blastocyst yield, hatching rate and ICM/total cell number were similar in both fast and slow developing embryos. Therefore, based on the results of the other studies, one may consider that while the fast developing embryos produce blastocysts with higher cryosurvival rate, the step-wise kinetics of the early embryo development have no effect on cryosurvival and developmental competence of these embryos. However, the exact reason of this phenomenon remains to be elucidated.
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Concluding remarks The results of the present study indicated that the optimal stage of early embryo vitrification is post-EGA stage. However, this study did not find a significant effect of kinetics of embryo development at and after EGA on cryosurvival of in vitro bovine embryos. In conclusion, the results of this study may provide a platform to develop better strategies for selection of the optimal stage of embryo development for vitrification. However, further studies can be conducted for comparative analysis of the transcriptomics, proteomics, and metabolomics of the early embryos before and after vitrification–warming. Author contributions A.V., H.S.M., and N.M.H., conceived and designed the experiments. A.V., H.S.M., F.M., and H.M., performed the experiments. A.V., and H.S.M., analyzed data. A.V., H.S.M., and N.M.H., wrote the paper. Acknowledgments This study was funded by a Grant from Royan Institute of IRI. The authors would like to thank Mrs. Mansouri for statistical analysis. The authors are also grateful to Mr. Heidari and Mr. Khajeh for ovaries preparation. References [1] M. Al-Hasani, M. Ludwig, F. Gagsteiger, W. Kupker, R. Stum, A. Yimaz, O. Bauer, K. Diedrich, Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intra-cytoplasmic sperm injection and conventional in vitro fertilization, Hum. Reprod. 11 (1996) 604–607. [2] D.H. Betts, W.A. King, Genetic regulation of embryo death and senescence, Theriogenology 55 (2001) 171–191. [3] E. Bianchi, C. Sette, Post-transcriptional control of gene expression in mouse early embryo development: a view from the tip of the Iceberg, Genes 2 (2011) 345–359. [4] M.R. Blanco, S. Demyda, M. Moreno Millán, E. Genero, Developmental competence of in vivo and in vitro matured oocytes: a review, Biotechnol. Mol. Biol. Rev. 6 (2011) 155–165. [5] D. Boonkusol, A.B. Gal, S. Bodo, B. Gorhony, Y. Kitiyanant, A. Dinnyes, Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos, Mol. Reprod. Dev. 73 (2006) 700–708. [6] X.S. Cui, N.H. Kim, Maternally derived transcripts: identification and characterization during oocyte maturation and early cleavage, Reprod. Fertil. Dev. 9 (2007) 25–34. [7] T.Q. Dang-Nguyen, K. Kikuchi, T. Somfai, M. Ozawa, M. Nakai, N. Maedomari, N. Viet-Linh, Y. Kanai, X. Nguyen, T. Nagai, Evaluation of developmental competence of in vitro-produced porcine embryos based on the timing, pattern and evenness of the first cleavage and onset of the second cleavage, J. Reprod. Dev. 56 (2010) 593–600. [8] A. Dhali, V.M. Anchamparuthy, S.P. Butler, R.E. Pearson, I.K. Mullarky, F.C. Gwazdauskas, Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos, Theriogenology 71 (2009) 1408–1416. [9] A. Dinnyés, P. Lonergan, T. Fair, M.P. Boland, X. Yang, Timing of the first cleavage post-insemination affects cryosurvival of in vitro-produced bovine blastocysts, Mol. Reprod. Dev. 53 (1999) 318–324. [10] M. Hajian, S.M. Hosseini, V. Asgari, S. Ostadhosseini, M. Forouzanfar, M.H. Nasr Esfahani, Effect of culture system on developmental competence, cryosurvival and DNA-fragmentation of in vitro bovine blastocysts, IJFS 5 (2011) 21–26. [11] S.M. Hosseini, F. Moulavi, M. Hajian, P. Abedi, M. Forouzanfar, S. OstadHosseini, L. Hosseini, A. Pirestani, H. Ghasemzadeh Nava, P. Tajik, A.H. Shahverdi, M.H. Nasr-Esfahani, Highly efficient in vitro production of bovine blastocyst in cellfree sequential synthetic oviductal fluid vs. TCM199 vero cell co-culture system, IJFS 2 (2008) 66–73. [12] S. Kattera, P. Shristav, I. Craft, Comparison of pregnancy outcome of pronuclear and multicellular stage frozen–thawed embryo transfers, J. Assist. Reprod. Genet. 16 (1999) 358–362.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics q V. Asgari a, S.M. Hosseini a, M. Forouzanfar b, M. Hajian a, M.H. Nasr-Esfahani a,c,⇑ a
Department of Reproduction and Development, Reproductive Biomedicine Centre, Royan Institute of Biotechnology, ACECR, Isfahan, Iran Department of Basic Science, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran c Department of Embryology, Reproductive Biomedicine Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran b
a r t i c l e
i n f o
Article history: Received 18 February 2012 Accepted 8 August 2012 Available online 21 August 2012 Keywords: Bovine Embryo Vitrification Embryonic genome activation Kinetics
a b s t r a c t In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8–16 cell stage): I and II – early (day 2) and late (day 3) 5–8 cell, III and IV – early (day 3) and late (day 4) 8–16 cell, and V and VI – early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5–8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8–16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Successful cryopreservation of embryos was first reported in 1971 in mice [36]. Increased possibility of embryo transfer technology in domestic animals and then human resulted in a parallel growing focus of interest to cryopreserve embryos in these species [27,37]. However, one dilemma that has remained to be addressed is the optimal embryonic stage (early stage vs. blastocyst) at which the embryos are preserved [22]. Theoretically, blastocyst stage embryos have overcome the embryonic block and a first selection has already been made. These embryos have greater nuclear cytoplasmic ratio which makes them more suitable for cryopreservation [20]. The embryo developmental block is a phenomenon that occurs in many species at different stages (for example third to fourth cell Abbreviations: EGA, embryonic genome activation; COC, cumulus oocyte complex; VS, vitrification solution; Me2SO, dimethyl sulfoxide; EG, ethylene glycol; RT, room temperature; ES, equilibration solution; TCN, total cell number; ICM, inner cell mass; TE, trophectoderm. q Statement of funding: This study was performed by the Grant of Royan Institute. There is no conflict of interest in this study. ⇑ Corresponding author at: Department of Reproduction and Development, Reproductive Biomedicine Center, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran. Fax: + 98 311 2605525. E-mail addresses: [email protected], [email protected] (M.H. Nasr-Esfahani).
0011-2240/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2012.08.002
cycle in humans, second cell cycle in mice and fourth or between the fourth and fifth cell cycle transition in bovine) due to the inability to activate zygotic genes and continue cleavage [30]. This species-specific block moment is associated with the developmental stage when embryos have to rely on the mRNAs transcribed from its own genome to continue cleavage [21]. In practice, the rate of ‘‘take home baby rate’’ per vitrified– warmed embryo at blastocyst stage is higher than embryos which are vitrified at earlier stages of development [22]. However, considering the potential disadvantages of prolonged in vitro embryo culture, there is an indispensable focus on vitrification of early stages embryos [4,22,24]. Although vitrification of early stage has been a matter of intensive focus in recent years, the impact of embryonic block and developmental kinetics (timing of first and subsequent embryonic cleavage) has been less noticed. It is well established that early embryonic events are orchestrated through the post-transcriptional control of maternal mRNA that have been accumulated during oogenesis [31]. These maternal mRNA and protein serve the distinct purpose of reprogramming and facilitating the early mitotic divisions of the fertilized embryos until broad embryonic genome activation (EGA) that is initiated at a species-specific stage of embryo development [3,6], so that the precise control over the time of EGA is essential for normal embryogenesis. Importantly, a significant reduction in mRNA content of vitrified–warmed early embryos has been documented in
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different species [5,32]. Therefore, considering the critical importance of maternally reserved mRNA for early embryo development, it is vital to determine if vitrification of embryos before or after EGA may affect cryosurvival of embryos. This approach requires an animal model with late embryonic genome activation. It is also well established that the time of the first cleavage postinsemination has major, long-lasting effects on subsequent development of embryos [14]. The rate of blastocyst development and the cell number of the resulting embryos have been related to the time of the first cleavage [14,7]. The evidences from both human and non-human studies suggest that early-cleaved embryos produced blastocysts, are more likely to survive cryopreservation and establishment pregnancies following embryo transfer than blastocyst derived from late cleaved embryos [22,28,38]. However, it is not clearly comprehended that how the step-wise kinetics of embryo development during different stages of preimplantation may affect cryosurvival of the embryos. Such studies would lead, in practice, to design more efficient strategies for selection the optimal stage of embryos development for vitrification. Therefore, the objective of the present study was to investigate the effect of developmental stage and kinetics of embryo development on cryosurvival of in vitro produced bovine embryos. Materials and methods Unless otherwise stated, all chemicals and media were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Gibco (Grand Island, NY, USA), respectively. In vitro oocyte maturation The process of oocyte IVM was as described previously [23]. In brief, ovaries from adult cows obtained from a local slaughterhouse, placed in saline (35 °C) and transported to the laboratory
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within 3–5 h. Cumulus–oocyte complexes (COCs) were aspirated from antral follicles (2–8 mm in diameter) using 18-gauge needles attached to vacuum pump (80 mmHg). COCs with homogenous cytoplasm and more than three layers of cumulus cells were then matured in tissue culture medium 199 (TCM199) containing 2.5 mM Na-pyruvate, 100 IU/ml penicillin, 100 lg/ml streptomycin, 1 mg/ml estradiol-17b, 10 lg/ml FSH, 10 lg/ml LH, 100 ng/ml EGF, 0.1 mM cysteamine and 10% fetal calf serum (FCS) at 39 °C in a humidified atmosphere containing 5% CO2 (C200, Labotect, Germany). Sperm preparation and in vitro embryo development Frozen–thawed and washed sperm from a single Holstein sire of proven in vitro fertility were used for fertilization after capacitation by the swim-up procedure. Spermatozoa (1 106 sperms/ ml) and matured COCs (40–45 COCs/200 ll) were co-incubated in modified fert-TALP medium containing 0.01 mM heparin, 0.2 mM penicillamine, and 0.1 mM hypotaurine at 38.5 °C for 18– 24 h under 5% CO2 in humidified air. The presumptive zygotes were then vortexed for 90 s in Hepes-TALP to remove the cumulus cells. After that, they were washed once in H-TCM199 and twice in synthetic oviductal fluid (SOF) and then cultured as described above. Incubation was performed at 38.5 °C, under 5% CO2 and 5% O2 in humidified air [11]. Experimental design From day 3 onwards, cleaved embryos at different stages of development were sorted based on the kinetics of embryo development. Accordingly, early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. Early and late embryos at each stage were used for vitrification and control
Fig. 1. Diagrammatic representation of experimental design. Cleaved embryos at early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. Early and late embryos at each stage were used for vitrification and control experiments. E: early, L: late, UN: un-vitrified, V: vitrified. Bar represents 50 lm.
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experiments. After that, the vitrified embryos were immediately warmed and cultured along with their corresponding control groups until the blastocyst stage (Fig. 1). Vitrification of embryos The vitrification protocol used in this study was as described previously [10]. Briefly, embryos were pre-equilibrated in a solution of 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (Me2SO) in phosphate buffer saline free of calcium and magnesium (PBS ) supplemented with 20% FCS until the expanded blastocysts endured a period of shrinkage and returning (up to 8 min, at 38.5 °C). Equilibrated embryos were then exposed to vitrification solution consisting of 15% EG + 15% Me2SO + 0.5 M sucrose in holding medium at room temperature for 1 min, and then were loaded on the tips of the cryotops (Cryologic; CVM™, Fibreplug & Sleeve, Australia) with the minimum amount of vitrification solution and immediately plunged into the liquid nitrogen (LN2). For warming, embryos were removed from LN2 and quickly exposed into PBS + 20% FCS supplemented with 1 M sucrose for 1 min on a warm plate (38.5 °C). Then, embryos were transferred to PBS + 20% FCS containing 0.5 M sucrose for 3 min and finally washed thoroughly in PBS + 20% FCS before being cultured in SOF medium.
calculation of blastocyst formation rate is increased. Accordingly, although the potential of 5–8 cell stage embryos to survive vitrification and to further develop to the blastocyst stage was insignificantly lower than its corresponding control (un-vitrified 5–8 cell stage embryos), but it was significantly lower than vitrified, un-vitrified 8–16 cell and morula stage embryos (Fig. 2A), while these differences were not considerable for un-vitrified 5–8 cell stage embryos. Neither the vitrification process nor the stage of embryo development at the time of vitrification affected the hatching ability of the developed blastocysts (Fig. 2B). Moreover, differential analysis of ICM and TE did not reveal any significant difference between the vitrified and un-vitrified or the stage at which the embryos were frozen (Fig. 2C). Effect of developmental kinetics on cryosurvival Within each developmental stage, neither vitrification–warming nor the kinetics of embryo development affected their survival
Differential embryo staining for quality assessment In order to determine the total cell number (TCN) and the sole number of cells allocated in the sites of inner cell mass (ICM) and trophectoderm (TE), the hatched blastocysts in both groups were assigned to differential staining as described elsewhere [23]. In brief, hatched blastocysts were incubated in 500 ll of 1% Triton X-100 and 100 lg/ml propidium iodide (solution 1) for up to 30 s, depending on the size of the embryos, and then immediately transferred into a 500 ll solution of 100% ethanol plus 25 lg/ml Hoechst 33258 (solution 2). Care was taken to carry the minimum amount of solution 1 when the embryos were transferred into solution 2. The embryos were extensively washed in solution 2 to remove any trace amounts of solution 1. Samples were then stored in solution 2 at 4 °C overnight. Fixed and stained embryos were subsequently mounted onto a glass slide in one drop of glycerol, gently flattened with a cover slip and visualized for cell counting on a fluorescence microscope (excitation filter 460 nm for blue and 560 nm for red). TE cells were visualized as blue and ICM as pink to red. TCN was calculated by counting the numbers of both ICM and TE. Statistical analysis All of the experiments in the present study were repeated at least three times. Percentages data were modeled to the binomial model of parameters by ArcSin transformation. The transformed data were analyzed by one way ANOVA model of SPSS 17. Also, differences were compared by Tukey multiple comparison post hoc test. It is noteworthy to mention that all data were presented as means ± SEM and differences considered significant at P 6 0.05. Results Effect of developmental stage on cryosurvival Fig. 2 represents the effect of stage of embryo development on cryosurvival of the resultant embryos. As shown, irrespective of the vitrification procedure, developmental competence of embryos was increased as the developmental stage taken into account for
Fig. 2. Effect of developmental stage and vitrification on blastocyst formation, hatching rate and the number of inner cell mass (ICM) cells per total cells of bovine in vitro produced (IVP) embryos. V: vitrified, UV: un-vitrified. Different letters display significant differences at P 6 0.05.
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Fig. 3. Effect of developmental stage and vitrification on the kinetics of blastocyst formation of bovine IVP embryos (early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively). V: vitrified, UV: un-vitrified. Different letters display significant differences at P 6 0.05.
and subsequent potential to develop to the blastocyst stage (Fig. 3). Furthermore, the hatching ability (Fig. 4) and differential analysis of ICM and TE (Fig. 5) did not reveal any significant difference between vitrified and un-vitrified embryos. Moreover, differential allocations of ICM and TCN were not affected by developmental kinetics, irrespective of the day at which the blastocysts were formed. Discussion Literature study clearly revealed that pregnancy rate is substantially higher when blastocyst is vitrified–warmed and then transferred compared to transfer of vitrified–warmed early stage embryos [1,12,13,25,29,33]. There are several important reasons
Fig. 4. Effect of developmental stage and vitrification on the kinetics of hatching rate of bovine early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. V: vitrified, UV: un-vitrified.
for this difference, including higher cell number and increased nucleo-to-cytoplasmic ratio in blastocysts which guarantees optimum equilibrium with the cryoprotectants and allows an embryo recovery even if some cells are destroyed in response to cryodamages [22,16]. Another stark difference between blastocysts and early stage embryos is that the blastocysts have already overcome the species-specific genomic activation block and a first selection has already been made between developing and
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Fig. 5. Effect of developmental stage and vitrification on the number of inner cell mass (ICM) cells per total cell number (TCN) of resultant blastocyst (at day 8 and 9) of bovine early 5–8, 8–16 cell, and morula embryos were selected at days 2, 3 and 4 and late embryos of the same stages were selected at days 3, 4 and 5, respectively. V: vitrified, UV: un-vitrified.
non-developing embryos [20]. Despite the latter facts, the link between developmental kinetics, time of embryonic genome activation and cryosurvival of early bovine embryos remains to be defined. The results of this study showed that developmental stage had a deterministic impact on developmental competency of embryos which have survived vitrification to produced blastocyst in vitro. Accordingly, considering the time of embryo genome activation which corresponded to around 16 cell stage in bovine, it was ob-
served that vitrification of embryos before EGA (at 5–8 cell stage) significantly reduced their developmental competence compared to those embryos that were vitrified–warmed at the later stages or beyond EGA (8–16 cell or the morula stage). However, it is important to note that un-vitrified embryos at 5–8 cell stage did not show a significant reduction in the developmental competence as compared with later stage embryos, whereas, interestingly the majority of embryos that were vitrified–warmed at 5–8 cell stage arrested before morula stage. Two main mechanisms may describe the aforementioned difference: (i) inability to overcome the chromatin repression and activate transcription of important developmental genes and/or (ii) to react to injuries caused by environment [2], such as vitrification–warming. Cryopreservation method can be influence the developmental competency of vitrified–warmed embryos at different developmental stage. In this regard, Mandelbaum et al. [19] showed that optimal survival rates are only achieved when sucrose is combined with propanediol (PrOH) and propanediol itself leads to reduced survival rates. On the other hand, Van der Elst et al. [34] demonstrated that vitrification of multicellular embryos could be improved with DMSO. However, the success rates reported with the PrOH protocol were considerably low. The reduction of the developmental competence of vitrified vs. non-vitrified 5–8 cell stage embryos in this study can be described from two different perspectives; cellular and molecular. From cellular point of view, it is well established that the embryonic divisions is concomitant with a reduction in cellular volume [15] and it provides a better equilibration of the embryos with cryoprotectants, and thereby better cryo-withstand. Furthermore, embryo progression results in a subsequent increase in nucleo-to-cytoplasmic ratio which is beneficial to cryopreservation [16]. From the molecular point of view, vitrification of embryos at earlier stages of development before EGA may deprive embryos of maternal reserve of mRNAs that are responsible for completion of embryo development before EGA initiation. It is interesting that mouse embryos vitrified at two cell stage (time of EGA in mice) have had better cryosurvival rate than those vitrified before EGA (at zygotic stage) [8]. However, to provide a better understanding of the impact of the mentioned mechanisms, comparative analysis of the transcriptomics, proteomics, and metabolomics of the early embryos before and after vitrification–warming are beneficial. Kinetics of early embryo development is another important embryo characteristic which allows prediction of the potential of embryos to develop towards blastocyst stage [17]. Accordingly, it has been demonstrated that the rate of blastocyst development and the cell number of the resulting embryos correlate with the time of the first cleavage [35,18]. It has been also shown that timing of the first cleavage post-insemination can influence the cryosurvival of bovine blastocysts following vitrification [9]. Moreover, it has been indicated that fast developing embryos (expanded blastocyst and early hatching blastocyst stage) had better resistance to freezing than delayed ones (early blastocyst stage) derived from the same group of embryos which were inseminated concomitantly [26]. However, it was not clear if kinetics of embryo development beyond two cells stage can also affect cryosurvival and subsequent development. The results of this study indicated that for each developmental stage, kinetics of embryo development had no predictive value for embryos further development. Accordingly, all the parameters analyzed in this study including blastocyst yield, hatching rate and ICM/total cell number were similar in both fast and slow developing embryos. Therefore, based on the results of the other studies, one may consider that while the fast developing embryos produce blastocysts with higher cryosurvival rate, the step-wise kinetics of the early embryo development have no effect on cryosurvival and developmental competence of these embryos. However, the exact reason of this phenomenon remains to be elucidated.
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Concluding remarks The results of the present study indicated that the optimal stage of early embryo vitrification is post-EGA stage. However, this study did not find a significant effect of kinetics of embryo development at and after EGA on cryosurvival of in vitro bovine embryos. In conclusion, the results of this study may provide a platform to develop better strategies for selection of the optimal stage of embryo development for vitrification. However, further studies can be conducted for comparative analysis of the transcriptomics, proteomics, and metabolomics of the early embryos before and after vitrification–warming. Author contributions A.V., H.S.M., and N.M.H., conceived and designed the experiments. A.V., H.S.M., F.M., and H.M., performed the experiments. A.V., and H.S.M., analyzed data. A.V., H.S.M., and N.M.H., wrote the paper. Acknowledgments This study was funded by a Grant from Royan Institute of IRI. The authors would like to thank Mrs. Mansouri for statistical analysis. The authors are also grateful to Mr. Heidari and Mr. Khajeh for ovaries preparation. References [1] M. Al-Hasani, M. Ludwig, F. Gagsteiger, W. Kupker, R. Stum, A. Yimaz, O. Bauer, K. Diedrich, Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intra-cytoplasmic sperm injection and conventional in vitro fertilization, Hum. Reprod. 11 (1996) 604–607. [2] D.H. Betts, W.A. King, Genetic regulation of embryo death and senescence, Theriogenology 55 (2001) 171–191. [3] E. Bianchi, C. Sette, Post-transcriptional control of gene expression in mouse early embryo development: a view from the tip of the Iceberg, Genes 2 (2011) 345–359. [4] M.R. Blanco, S. Demyda, M. Moreno Millán, E. Genero, Developmental competence of in vivo and in vitro matured oocytes: a review, Biotechnol. Mol. Biol. Rev. 6 (2011) 155–165. [5] D. Boonkusol, A.B. Gal, S. Bodo, B. Gorhony, Y. Kitiyanant, A. Dinnyes, Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos, Mol. Reprod. Dev. 73 (2006) 700–708. [6] X.S. Cui, N.H. Kim, Maternally derived transcripts: identification and characterization during oocyte maturation and early cleavage, Reprod. Fertil. Dev. 9 (2007) 25–34. [7] T.Q. Dang-Nguyen, K. Kikuchi, T. Somfai, M. Ozawa, M. Nakai, N. Maedomari, N. Viet-Linh, Y. Kanai, X. Nguyen, T. Nagai, Evaluation of developmental competence of in vitro-produced porcine embryos based on the timing, pattern and evenness of the first cleavage and onset of the second cleavage, J. Reprod. Dev. 56 (2010) 593–600. [8] A. Dhali, V.M. Anchamparuthy, S.P. Butler, R.E. Pearson, I.K. Mullarky, F.C. Gwazdauskas, Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos, Theriogenology 71 (2009) 1408–1416. [9] A. Dinnyés, P. Lonergan, T. Fair, M.P. Boland, X. Yang, Timing of the first cleavage post-insemination affects cryosurvival of in vitro-produced bovine blastocysts, Mol. Reprod. Dev. 53 (1999) 318–324. [10] M. Hajian, S.M. Hosseini, V. Asgari, S. Ostadhosseini, M. Forouzanfar, M.H. Nasr Esfahani, Effect of culture system on developmental competence, cryosurvival and DNA-fragmentation of in vitro bovine blastocysts, IJFS 5 (2011) 21–26. [11] S.M. Hosseini, F. Moulavi, M. Hajian, P. Abedi, M. Forouzanfar, S. OstadHosseini, L. Hosseini, A. Pirestani, H. Ghasemzadeh Nava, P. Tajik, A.H. Shahverdi, M.H. Nasr-Esfahani, Highly efficient in vitro production of bovine blastocyst in cellfree sequential synthetic oviductal fluid vs. TCM199 vero cell co-culture system, IJFS 2 (2008) 66–73. [12] S. Kattera, P. Shristav, I. Craft, Comparison of pregnancy outcome of pronuclear and multicellular stage frozen–thawed embryo transfers, J. Assist. Reprod. Genet. 16 (1999) 358–362.
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