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Synchronization of cell division in eight-cell bovine embryos produced in vitro: Effects of aphidicolin
ELSEVIER
SYNCHRONIZATION
OF CELL DMSION IN EIGHT.-CELL BOVINE EMBRYOS PRODUCED IN VITRO: EFFECTS OF APHIDICOLIN S. Samake and L.C. Smitha
Cerme de mchetche en reproduction animale Departement de biom&cine veterinaire Fact& de m&k&e v&inaim Universitk de Mont&l, Saint-Hyacinthe, PQ, Canada J2S 7C6 Receivedforpublication: Accepted:
March 14, 1996 June 10, 1997
ABSTRACT To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomem division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 @vI and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos. 0 1997 by Elsevier Science Inc Key words: aphidicolin, cell cycle arrest, synchronization, bovine, embryo INTRODUCTION In contrast to amphibians, in which the transition from synchronous to asynchronous types of division occurs at the mid-blastula stage, mammalian blastomems divide at different times as early as the second cleavage, and this asynchrony increases among blastomeres throughout preimplantation development (32). To support a possible role for asynchronous cleavage in early differentiation events are studies showing that descendents of the earlier-dividing 2cell blastomem contribute significantly more to the inner cell mass of blastocysts (15,31). Moreover, cell cycle stage-diversity of blastomeres caused by cleavage asynchrony is believed to cause inconsistent nucleo-cytoplasmic interactions during oocyte reconstruction, leading to poor development in several mammalian species, including mice (6,18,26,27), rabbit (7,8), sheep (4) and cattle (2,5,30). Therefore, for both fundamental and practical reasons, it was deemed important to determine the effectiveness of different cell cycle-arresting agents that cause minimal effects to normal development. Acknowledgments The authors thank Carmen Ikveilk and Luc Moquin for excellent technical assistance, David McLay and Dr. Hugh Clarke, McGill University, for help with DNA synthesis technique; and Dr. A.K. Goff for aid in data analysis. Funded by CORPAQ and NSERC of Canada. a Correspondence and reprint request. Theriogenology 48:969-976, 1997 0 1997 by Elsewer Science Inc.
0093-691X/97/$17.00 PII SOO93-691X(97)00323-3
Theriogenology
970
Whereas several studies have reported on the effects of agents that arrest blastomem cell cycle at mitosis, i.e., nocodazole (4,18,22), few have attempted synchronization during interphase. Nonetheless, many DNA replication inhibitors have been used to arrest and synchronize actively dividing cells in the S-phase for nuclear transfer procedures. Among them, hydroxyutea has been used to study nuclear cytoplasmic interactions during the first cell cycle of reconstructed bovine embryos (5), while 5-fluordeoxyuridme (5,6,13), and mimosine (19) have been used to synchronize mouse embryos in the S-phase. The calmodulin antagonist W-7 is also a DNA replication inhibitor used to arrest mouse blastomems in S-phase (12,21). Aphidicolii, a powerful inhibitor of DNA polymerasc alpha and nuclear DNA replication, has become the agent of choice to study differentiation without cleavage of the Chaetopterus egg (l), DNA replication and the control of cell cycle in Xenopus egg extracts (14). Other studies have used aphidicolin to determine the relationship between the number of DNA replication cycles and the occurrence of mouse blastocyst formation (1,10,13,28,29,34), to analyze the role of DNA replication on changes of chromatin structure (25.35) and to synchronize rabbit embryos in Gl phase prior to nuclear transfer procedures (7). The efficiency of aphidicolin as a blastomete synchronizing agent has been compared to other drugs such as nocodazole and 6-DMAP during mouse early embryogenesis (22). In bovine embryos, although aphidicolm has been used to synchronize fertilized oocytcs in early S phase (1 l), no data are available on effects of this agent on cell cycle arrest and synchronization at cleavage stages of preimplantation development. Therefore, the aim of this study was to determine the lowest concentration of aphidicolin necessary to arrest cleavage in g-cell bovine embryos and to establish the reversibility and viability of treated embryos to develop normally to the blastocyst stage after treatment. MATE&US
AND METHODS
Embryo Source and Culture Bovine ovaries were collected at a local abattoir, and non-haemorrhagic follicles with a diameter of 1 to 5 mm were punctured with a 18-gauge needle to collect cumulus-cocyte complexes (CGCs). Groups of 20 CGCs with complete cumulus layers and homogenous ooplasm were matured for 24 h in 50 pl of TCM 199 bicarbonate buffeted medium (Gibco, Grand Island, NY) supplemented with 10 mg LH ml-t, 1 mg FSH ml-l, 2 ng estradiol ml-l and 10% FCS (G&o). At the end of maturation, expanded CGCs were fertilized by standard protocols (20). Briefly, matured oocytes were transferred to TALP medium containing 10 mg heparin ml-l and inseminated with frozen-thawed, Percoll gradient-separated spermatozoa (1 x 106 sperm ml-l). At 18 h post insemination (hpi), zygotes were transferred to 50-pl drops of INBA Menezo’s B2 medium (MB2; Pharmascience, Paris, France) with bovine oviduct epithelial cells (BOEC) for co-cultme. Eight-cell stage embryos obtained at 58 h post insemination were selected to test the ability of aphidicolin to armst cleavage. Eight-cell stage is the most advanced cleavage stage in which blastomem number can be precisely ascertained by microscopic observation. Further, to assess the reversibility of aphidicolin treatment, embryos were washed 3 times in Hepes-buffered TCM-199 and transferred to aphidicolin-free MB2 medium, and their ability to resume cleavage to the fifth cell cycle was determined. They were then assayed, during a period of several hours after release from aphidicolin, to determine the percentage of metaphase nuclei as an indication of timing and synchrony of mitotic division and the pattern of DNA synthesis. To assess toxicity of treatment, embryos were washed 3 times in Hepes-buffered TCM-199 followed by co-culture for a further 6 d in aphidicolin-free MB2 medium, and their ability to resume cleavage and develop into normal blastocysts in vitro was determined. Cleavage Arrest and Chromatin Morphology Assay Aphidicolin (Sigma, St-Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) (Sigma) at 1 mg ml-*. In preliminary experiments using similar 8-cell stage embryos, 12 h incubation periods in culture medium containing DMSO at up to 0.2% (v/v) had no effect on further development to the blastocyst stage. Working solutions were prepared by appropriate dilution of the stock into TCM-199
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medium conditioned with BOEC. Embryos were treated with concentrations ranging from 0 to 2.8 pM of aphidicolin for 12 h to determine the lowest effective dose to cause complete development arrest. At the end of treatment, embryos were evaluated for cleavage and chromatin morphology using a rapid staining procedure for nuclei. Briefly, embryos were fixed in 10% formahn and mounted onto a glass slide in a mounting solution (Mowiol; Aldrich, Milwaukee, WI) containing the DNA-specific dye bisbenzimide at 1 pg ml-l (Hoechst 33342; Sigma). Nuclear morphology and number of nuclei per embryo were determined with an inverted microscope equipped with epifluomscence (TMD-Diaphot, Nikon) and an ultraviolet filter block (330 to 380 nm excitation and 420 nm emission, UV-2A, Nikon). DNA Synthesis Assay After release from aphidicolin, groups of g-cell stage embryos were incubated for 1 h in MB2 medium supplemented with 100 pM of bromodeoxyuridine (BrdU; 5-bromo-2’ deoxyuridine Striphosphate, Sigma) at intervals of 2 h to determine the onset of DNA synthesis over the 10 h period after release. At the end of each incubation period, embryos were fvred in 10% formalin for 15 min. Plasma membranes were permeabilized in PBS 0.1% niton X-100 (Biopharm, Laval, Canada) for 15 min, and embryos were washed in block solution (PBS 3% BSA 0.1% Tween-20) for 30 min at room temperature. The embryos were then incubated for 1 hr in a humidified chamber with 10 p.l antiBrdU moncclonal antibody (Amersham) containing DNAse 1 pg ml-l. After incubation, embryos were washed twice in block solution for 10 min and incubated for 1 h in 10 p.l of fluomscein @TAF)conjugated affinity purified goat anti-mouse IgG (Sigma) at 1:lOO dilution. The embryos were then washed twice in block solution for 10 min, mounted onto slides in Mowiol containing Hoechst 33342 1 pg ml-l and examined by epifluomscence. Percentages were analyzed by Chi-square and blastocyst cell numbers by ANOVA. RESULTS Complete arrest of cleavage was induced by aphidicolin in these studies. The effects of this agent with respect to dose mquimd for arrest, reversibility and toxicity are described below. Cell-cycle Arrest Embryos were considered arrested when containing 8 nuclei and non-arrested when the number of nuclei was mom than 8 at the end of treatment. A total of 200 embryos, 20 embryos per concentration, was exposed for 12 h to concentrations of aphidicolin ranging from 0 to 2.8 pM, after which they were fixed to assess cleavage arrest and chromatin morphology. Control and 0.35 pMtreatment had no effect on cleavage. In the control group, 90% of embryos had cleaved, i.e., had more than 8 cells within 6 h after exposure of the treated embryos to aphidicolin (P
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035 0.70 1.40 Concentration of aphidicolin Q.&l) Figure 1
2.80
The ability of aphidicobn to induce in vitro cleavage arrest at concentrations of 0 to 2.8 pM in 8-tell bovine embryos. N=60 for each column.
Figure 2 Chromatin morphology of 8-tell bovine embryos, (c) after 1Zhour exposure to 1.4 pM of aphidicolin compared to (a) controls at the moment of exposure (58 hpi) and, (b) 12 hours later (70 hpi). Bar=100 pm removed from aphidicolin and assayed for BrdU incorporation at 2-h intervals for the following 10 h (Figure 3b). At the end of treatment and 2 h after release from aphidicolin, less than 1% of blastomeres were incorporating BrdU, thus indicating that DNA synthesis was inhibited in 99% of blastomeres (P&05). Levels of BrdU incorporation increased to 38% by 4 h and kept increasing to reach a maximum of 55% by 8 h, followed by a decrease to 12% by 10 h after removal of aphidicolin (PcO.05). The percentage of blastomexe nuclei presenting positive staining for BrdU oscillated between 3 and 58% during the same period in the control embryos.
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1 Tie
3
4
0
2
4
6
8
10
from release from aphidicolin arrest (hours)
Figure 3 Cell cycle reversibility of aphidicolin-treated bovine embryos. (a) Percentage of nuclei found in me&phase at several periods after removal of aphidicolin (closed circles) compared with controls (open circles), and (b) percentage of blastomems positively showing BrdU incorporation in tmated embryos several hr after removal of aphidicolin compared with controlsVertical bars indicate the standard error among replicates. Toxicity To assess the toxicity of aphidicolin on embryo development in vitro, 180 eight-cell control and treated embryos exposed for 12 h to concentrations of aphidicolin ranging from 0.7 to 2.8 pM were washed and cultured in aphidicolin-free MB2 medium for a further 6 d (Figure 4). Concentrations above 0.7 l.tM used in this study, including the minimal effective dose of 1.4 )IM, had a significant effect on the ability of treated embryos to develop to the blastocyst stage. At these concentrations, a significant effect was also observed on the number of cells per blastocyst when compared with the controls (P&.05). Further, the hatching rate of blastocysts at Day 6 of culture was also affected when using the minimal effective dose of 1.4 pM as compared with that of the controls (P&.05; Figure 4). DISCUSSION Our results indicate that cleavage to the fifth cell cycle can be successfully arrested in 8-c&l bovine embryos with aphidicolin at 0.7 l&I. Moreover, resumption of DNA synthesis does not occur in treated embryos up to 4 h after removal from aphidicolin as assessed by BrdU incorporation. These results are supported by previous findings where complete cleavage arrest with aphidicolin was obtained with bovine &cell embryos exposed at 50 h but only partially at 74 h post insemination (25). Aphidicolin has been shown to act by specifically inhibiting DNA polymerase alpha, preventing DNA synthesis at the S phase of the cell cycle and, thereby, arresting embryos in interphase (29). In this study, less than 1% of blastomeres were incorporating BrdU at the end of treatment and up to 2 h after removal of aphidicolin, indicating the powerful effect of this agent on DNA polymerase alpha inhibition. Incorporation of BrdU increased by 4 h (38%) to reach a maximum of 55% by 8 h before decreasing to 12% 10 hr after removal from aphidicolin.
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100 P, 80 A B 60 B -40 3 e 20 8: 0
l(a)
PcO.05 I a
a
0.70 control 1.40 Concentration of aphidicolin (PM)
2.80
Figure 4 Effects of aphidicolin at doses of 0.7 to 2.8 pM on, (a) the in vitro development of g-cell bovine embryos to the blastocyst stage, (b) the hatching rate and, (c) the number of nuclei at Day 6after removal of the agent. Hatched bars indicate effects of aphidicolin at the minimal effective dose of 1.4 PM of aphidicolin. N=180 per experiment_ Different superscripts indicate 5% level of significance (P&05). Metaphase-like chromatin was observed in 43% of the blastomeres at the end of tmaunent, suggesting that aphidicolin may cause metaphase arrest in some g-cell stage blastometes. Although the mechanisms leading to the metaphase arrest observed in our studies am unknown, it is possible that aphidicolin induces irreversible chromatin condensation leading to cellular death. This assumption is supported by the stable percentage of metaphase nuclei present during several hours after release, suggesting that a percentage of nuclei are not re-entering the cell cycle. Further, lower cell numbers at the blastocyst stage may have resulted from this irreversible arrest of some blastomeres at the g-cell stage. Aphidicolin may affect the major transcriptional activation that occurs at the g-cell stage in cattle (3,16) and, in such way, lead to irreversible damage and blockage of cleavage to the fifth cell cycle. In mice, inhibition of DNA synthesis in Gl preceding transcriptional
Theriogenology
activation at the second cell cycle prevents later differentiation events regardless of the occurrence of a “catch-up” round of DNA synthesis immediately after the removal of the inhibitor (28). Observations on the inability of several treated embryos to develop to the blastocyst stage, a significant decrease in the number of cells per blastocyst and in the hatching rate, indicates that aphidicolin is detrimental when used for bovine S-cell stage blastomere synchronization. Toxicity to aphidicolin has been reported to be stage-dependent in mouse embryos, with toxic effects having been observed before (1,9,10,29) and after the 8-cell stage where aphidicolin treatment led to a deficiency in inner cell-mass (ICM) cell number in blastocysts (10,13,22,29). In the latter case, normal ICM development was obtained when cell number was increased by aggregating embryos during aphidicolin treatment, suggesting that this deficiency was due to the low cell number rather than the inability of ICM to form after exposure to the artest agent (10). In cattle, no data are available on stage-dependent developmental sensitivity to aphidicolin and it is yet unclear whether treatment of bovine embryos at other stages than 8-cell will be less detrimental to normal development, Although it is possible that, due to the activation of transcription, bovine 8-cell stage am particularly sensitive to exposure periods lasting 12 h, other cell cycle arrest-agents have shown little or no detrimental effects on development (23,24). Since the latter studies were able to induce a synchronous entry into the fifth cell cycle within 3 to 4 h after removal, it is possible that short-term exposures to aphidicolin following treatment with nocodazole or 6-Dimethylaminoprine would enable precise cell cycle arrest at the Gl to S-phase transition with fewer detrimental effects to development to the blastocyst stage. Similar approaches have been used with somatic cell lines using arrest at GO by serum starvation followed by the addition of serum in the presence of either aphidicolin (33) or mimosin (17) to arrest the cells at the GUS boundary. After removal from the DNA synthesis inhibitors, cells proceeded synchronously through the S and G2 phases, allowing manipulations at specific points in either phase. Further studies using multiple synchronization protocols are required to enable proper and reversible cell cycle arrest of cattle pteimplantation embryos. REFERENCES 1. Alexandre H. Effet de l’inhibition specitique de la replication de I’ADN par 1’Aphidicoline sur la differentiation primaim de l’oeuf de souris en preimplantation. Cons Rech Acad Sci Paris 1982; 294: 1001-1006. 2. Barnes FL, Collas P, Powell R, Ring WA, Westhusin M, Shepherd D. Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown. chromosome constitution, and development in nuclear transplant bovine embryos. Dev Biol 1993; 36: 33-41. 3. Camous S, Kopecny V, Flkhon J-E. Autoradiographic detection of the earliest stage of (3H)uridine incorporation into the cow embryo. Biol Cell 1986; 58: 195200. 4. Campbell KHS, Loi P, Cappai P, Wilmut I. Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol Reprod 1994; 50: 13851393. 5. Campbell RI-IS, Ritchie WA, Wilmut I. Nuclear-cytoplasmic interactions during the fust cell cycle of nuclear transfer reconstructed bovine embryos: Implications for deoxyribonucleic acid replication and development. Biol Reprod 1993; 49: 933-942. 6. Cheong I-IT, Takahashi Y, Kanagawa H. Birth of mice after transplantation of early cell cycle stage embryonic nuclei into enucleated oocytes. J Reprod Fertill993; 48: 958-963. 7. Collas P, Balk JJ, Rob1 JM. Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol Reprod 1992; 46: 492-500. 8. Collas P, Pinto-Correia C, DeLeon FAP, Rob1 JM. Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos. Biol Reprod 1992; 46: 501-511. 9. Cozad KM, Warner CM. The effects of aphidicolin and a-amanitin on DNA synthesis in preimplantation mouse embryos. Gamete Res 1982; 6: 155-160. 10. Dean WL, Rossant J. Effect of delaying DNA replication on blastocyst formation in the mouse. Differentiation 1984; 26: 134-137.
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11. Gag& M, Pothier F, Sirard M-A Effect of microinjection time during post-fertilization S-phase on bovine embryonic development Mol Reprod Dev 1995; 41: 184-194. 12. Hidaka H, Tanaka T. Naphthalenesulfonamides as cahnodulin inhibitors. Meth Enzym 1983; 102: 185194. 13. Howlett SK The effect of inhibiting DNA replication in the one-cell mouse embryo. Roux’s Arch Dev Biol1986; 195: 499-505. 14. Hutchmson CJ, Brill D, Cox R, Gilbert J, Kill I, Ford CC. DNA replication and cell cycle control in Xenopus egg extracts. J Cell Sci 1989; 12: 197-212. 15. Kelly SJ, Mulnard JG, Graham CF. Cell division and cell allocation in early mouse development. J Embryo1 Exp Morph01 1978; 48: 37-51. 16. Kopecny V, Flechon JE, Camous S, Fulka J,Jr.. Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine structural autoradiographic study. Mol Reprod Dev 1989; 1: 79-90. 17. Grren DK, Petersen LN, Bohr VA. A W-responsive G2 checkpoint in rodent cells. Mol Cell Biol 1995; 15: 3722-3730. 18. Gtaegui PJ, G’Neil GT, Campbell KHS, Wilmut I. Transfer of nuclei from 8-cell stage mouse embryos following use of Nocodazole to control the cell cycle. Mol Reprod Dev 1994; 39: 147152. 19. Ouhibi N, Fulka J,Jr., Kanka J, Moor RM. A reversible block at the GUS border during cell cycle progression of mouse embryos. Int J Dev Biol 1994; 38: 731-736. 20. Parrish JJ, Susko-Parrish JL, Leibfriedge-Ruthedge ML, Critser ES, Eyestone WH, First NL. Bovine in-vitro fertilization with frozen thawed semen. Theriogenology 1986; 25: 591-600. 21. Poueymirou WT, Schultz RM. Regulation of mouse pmirnplantation development: inhibitory effect of cahnodulin antagonist W-7 on the first cleavage. Mol Reprod Dev 1990; 26: 2 11-216. 22. Samake S, Smith LC. Effect of cell-cycle-arrest agents on cleavage and development of mouse embryos. J Exp Zoo1 1995; 274: 11 l-120. 23. Samake S, Smith LC. Synchronization of cell division in bovine embryos produced in vitro: effects of nocodazole. Mol Reprod Dev 1996; 44: 486-492. 24. &make S, Smith LC. Synchronization of cell division in eight-cell bovine embryos produced in vitro:effectsof 6-dimethyllaminopurine. J Reprod Fertil1997; 110: 21-27. 25. Smith LC, Meimlles FV, Bustin M, Clarke HJ. Assembly of somatic histone Hl onto chromatin during bovine early embryogenesis. J Exp Zoo1 1995; 273: 317-326. 26. Smith LC, Wilmut I, Hunter RHF. Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos. J Reprod Fertil 1988; 84: 619-624. 27. Smith LC. Wihnut I, West JD. Control of fast cleavage in single-cell reconstituted mouse embryos. J Reprod Fertil 1990; 88: 655-663. 28. Smith RKW, Johnson MH. DNA replication and compaction in the cleaving embryo of the mouse. J Embryo1 Exp Morph01 1985; 89: 133-148. 29. Spindle A, Nagano H, Pedersen RA. Inhibition of DNA replication in pmimplantation mouse embryos by aphidicolin. J Exp Zoo1 1985; 235: 289-295. 30. Stice SL, Keefer CL, Matthews L. Bovine nuclear transfer embryos: oocyte activation prior to blastomete fusion. Mol Reprod Dev 1994; 38: 61-68. 31. Surani MAH, Barton SC. Spatial distribution of blastomeres is dependent on cell division order and interactions in mouse mot&e. Dev Biol1984; 102: 335343. 32. Sutherland AE, Speed TP, Calarco PG. Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage. Dev Biol 1990, 137: 13-25. 33. Tobey RA, Valdez JG, Crissman HA. Synchronization of human diploid tibroblasts at multiple stages of the cell cycle. Exp Cell Res 1988; 179: 400-416. 34. Valdimarsson G, Kidder GM. Temporal control of gap junction assembly in preimplantation mouse embryos. J Cell Sci 1995; 108: 1715-1722. 35. Wormd DM, Turner BM, Schultz RM. Temporally mstricted spatial localization of acetylated isoforms of hitone H4 and RNA polymerasc II in the 2-tell mouse embryo. Development 1995; 121: 2949-2959.
SYNCHRONIZATION
OF CELL DMSION IN EIGHT.-CELL BOVINE EMBRYOS PRODUCED IN VITRO: EFFECTS OF APHIDICOLIN S. Samake and L.C. Smitha
Cerme de mchetche en reproduction animale Departement de biom&cine veterinaire Fact& de m&k&e v&inaim Universitk de Mont&l, Saint-Hyacinthe, PQ, Canada J2S 7C6 Receivedforpublication: Accepted:
March 14, 1996 June 10, 1997
ABSTRACT To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomem division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 @vI and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos. 0 1997 by Elsevier Science Inc Key words: aphidicolin, cell cycle arrest, synchronization, bovine, embryo INTRODUCTION In contrast to amphibians, in which the transition from synchronous to asynchronous types of division occurs at the mid-blastula stage, mammalian blastomems divide at different times as early as the second cleavage, and this asynchrony increases among blastomeres throughout preimplantation development (32). To support a possible role for asynchronous cleavage in early differentiation events are studies showing that descendents of the earlier-dividing 2cell blastomem contribute significantly more to the inner cell mass of blastocysts (15,31). Moreover, cell cycle stage-diversity of blastomeres caused by cleavage asynchrony is believed to cause inconsistent nucleo-cytoplasmic interactions during oocyte reconstruction, leading to poor development in several mammalian species, including mice (6,18,26,27), rabbit (7,8), sheep (4) and cattle (2,5,30). Therefore, for both fundamental and practical reasons, it was deemed important to determine the effectiveness of different cell cycle-arresting agents that cause minimal effects to normal development. Acknowledgments The authors thank Carmen Ikveilk and Luc Moquin for excellent technical assistance, David McLay and Dr. Hugh Clarke, McGill University, for help with DNA synthesis technique; and Dr. A.K. Goff for aid in data analysis. Funded by CORPAQ and NSERC of Canada. a Correspondence and reprint request. Theriogenology 48:969-976, 1997 0 1997 by Elsewer Science Inc.
0093-691X/97/$17.00 PII SOO93-691X(97)00323-3
Theriogenology
970
Whereas several studies have reported on the effects of agents that arrest blastomem cell cycle at mitosis, i.e., nocodazole (4,18,22), few have attempted synchronization during interphase. Nonetheless, many DNA replication inhibitors have been used to arrest and synchronize actively dividing cells in the S-phase for nuclear transfer procedures. Among them, hydroxyutea has been used to study nuclear cytoplasmic interactions during the first cell cycle of reconstructed bovine embryos (5), while 5-fluordeoxyuridme (5,6,13), and mimosine (19) have been used to synchronize mouse embryos in the S-phase. The calmodulin antagonist W-7 is also a DNA replication inhibitor used to arrest mouse blastomems in S-phase (12,21). Aphidicolii, a powerful inhibitor of DNA polymerasc alpha and nuclear DNA replication, has become the agent of choice to study differentiation without cleavage of the Chaetopterus egg (l), DNA replication and the control of cell cycle in Xenopus egg extracts (14). Other studies have used aphidicolin to determine the relationship between the number of DNA replication cycles and the occurrence of mouse blastocyst formation (1,10,13,28,29,34), to analyze the role of DNA replication on changes of chromatin structure (25.35) and to synchronize rabbit embryos in Gl phase prior to nuclear transfer procedures (7). The efficiency of aphidicolin as a blastomete synchronizing agent has been compared to other drugs such as nocodazole and 6-DMAP during mouse early embryogenesis (22). In bovine embryos, although aphidicolm has been used to synchronize fertilized oocytcs in early S phase (1 l), no data are available on effects of this agent on cell cycle arrest and synchronization at cleavage stages of preimplantation development. Therefore, the aim of this study was to determine the lowest concentration of aphidicolin necessary to arrest cleavage in g-cell bovine embryos and to establish the reversibility and viability of treated embryos to develop normally to the blastocyst stage after treatment. MATE&US
AND METHODS
Embryo Source and Culture Bovine ovaries were collected at a local abattoir, and non-haemorrhagic follicles with a diameter of 1 to 5 mm were punctured with a 18-gauge needle to collect cumulus-cocyte complexes (CGCs). Groups of 20 CGCs with complete cumulus layers and homogenous ooplasm were matured for 24 h in 50 pl of TCM 199 bicarbonate buffeted medium (Gibco, Grand Island, NY) supplemented with 10 mg LH ml-t, 1 mg FSH ml-l, 2 ng estradiol ml-l and 10% FCS (G&o). At the end of maturation, expanded CGCs were fertilized by standard protocols (20). Briefly, matured oocytes were transferred to TALP medium containing 10 mg heparin ml-l and inseminated with frozen-thawed, Percoll gradient-separated spermatozoa (1 x 106 sperm ml-l). At 18 h post insemination (hpi), zygotes were transferred to 50-pl drops of INBA Menezo’s B2 medium (MB2; Pharmascience, Paris, France) with bovine oviduct epithelial cells (BOEC) for co-cultme. Eight-cell stage embryos obtained at 58 h post insemination were selected to test the ability of aphidicolin to armst cleavage. Eight-cell stage is the most advanced cleavage stage in which blastomem number can be precisely ascertained by microscopic observation. Further, to assess the reversibility of aphidicolin treatment, embryos were washed 3 times in Hepes-buffered TCM-199 and transferred to aphidicolin-free MB2 medium, and their ability to resume cleavage to the fifth cell cycle was determined. They were then assayed, during a period of several hours after release from aphidicolin, to determine the percentage of metaphase nuclei as an indication of timing and synchrony of mitotic division and the pattern of DNA synthesis. To assess toxicity of treatment, embryos were washed 3 times in Hepes-buffered TCM-199 followed by co-culture for a further 6 d in aphidicolin-free MB2 medium, and their ability to resume cleavage and develop into normal blastocysts in vitro was determined. Cleavage Arrest and Chromatin Morphology Assay Aphidicolin (Sigma, St-Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) (Sigma) at 1 mg ml-*. In preliminary experiments using similar 8-cell stage embryos, 12 h incubation periods in culture medium containing DMSO at up to 0.2% (v/v) had no effect on further development to the blastocyst stage. Working solutions were prepared by appropriate dilution of the stock into TCM-199
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medium conditioned with BOEC. Embryos were treated with concentrations ranging from 0 to 2.8 pM of aphidicolin for 12 h to determine the lowest effective dose to cause complete development arrest. At the end of treatment, embryos were evaluated for cleavage and chromatin morphology using a rapid staining procedure for nuclei. Briefly, embryos were fixed in 10% formahn and mounted onto a glass slide in a mounting solution (Mowiol; Aldrich, Milwaukee, WI) containing the DNA-specific dye bisbenzimide at 1 pg ml-l (Hoechst 33342; Sigma). Nuclear morphology and number of nuclei per embryo were determined with an inverted microscope equipped with epifluomscence (TMD-Diaphot, Nikon) and an ultraviolet filter block (330 to 380 nm excitation and 420 nm emission, UV-2A, Nikon). DNA Synthesis Assay After release from aphidicolin, groups of g-cell stage embryos were incubated for 1 h in MB2 medium supplemented with 100 pM of bromodeoxyuridine (BrdU; 5-bromo-2’ deoxyuridine Striphosphate, Sigma) at intervals of 2 h to determine the onset of DNA synthesis over the 10 h period after release. At the end of each incubation period, embryos were fvred in 10% formalin for 15 min. Plasma membranes were permeabilized in PBS 0.1% niton X-100 (Biopharm, Laval, Canada) for 15 min, and embryos were washed in block solution (PBS 3% BSA 0.1% Tween-20) for 30 min at room temperature. The embryos were then incubated for 1 hr in a humidified chamber with 10 p.l antiBrdU moncclonal antibody (Amersham) containing DNAse 1 pg ml-l. After incubation, embryos were washed twice in block solution for 10 min and incubated for 1 h in 10 p.l of fluomscein @TAF)conjugated affinity purified goat anti-mouse IgG (Sigma) at 1:lOO dilution. The embryos were then washed twice in block solution for 10 min, mounted onto slides in Mowiol containing Hoechst 33342 1 pg ml-l and examined by epifluomscence. Percentages were analyzed by Chi-square and blastocyst cell numbers by ANOVA. RESULTS Complete arrest of cleavage was induced by aphidicolin in these studies. The effects of this agent with respect to dose mquimd for arrest, reversibility and toxicity are described below. Cell-cycle Arrest Embryos were considered arrested when containing 8 nuclei and non-arrested when the number of nuclei was mom than 8 at the end of treatment. A total of 200 embryos, 20 embryos per concentration, was exposed for 12 h to concentrations of aphidicolin ranging from 0 to 2.8 pM, after which they were fixed to assess cleavage arrest and chromatin morphology. Control and 0.35 pMtreatment had no effect on cleavage. In the control group, 90% of embryos had cleaved, i.e., had more than 8 cells within 6 h after exposure of the treated embryos to aphidicolin (P
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035 0.70 1.40 Concentration of aphidicolin Q.&l) Figure 1
2.80
The ability of aphidicobn to induce in vitro cleavage arrest at concentrations of 0 to 2.8 pM in 8-tell bovine embryos. N=60 for each column.
Figure 2 Chromatin morphology of 8-tell bovine embryos, (c) after 1Zhour exposure to 1.4 pM of aphidicolin compared to (a) controls at the moment of exposure (58 hpi) and, (b) 12 hours later (70 hpi). Bar=100 pm removed from aphidicolin and assayed for BrdU incorporation at 2-h intervals for the following 10 h (Figure 3b). At the end of treatment and 2 h after release from aphidicolin, less than 1% of blastomeres were incorporating BrdU, thus indicating that DNA synthesis was inhibited in 99% of blastomeres (P&05). Levels of BrdU incorporation increased to 38% by 4 h and kept increasing to reach a maximum of 55% by 8 h, followed by a decrease to 12% by 10 h after removal of aphidicolin (PcO.05). The percentage of blastomexe nuclei presenting positive staining for BrdU oscillated between 3 and 58% during the same period in the control embryos.
Theriogenology
0
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2
1 Tie
3
4
0
2
4
6
8
10
from release from aphidicolin arrest (hours)
Figure 3 Cell cycle reversibility of aphidicolin-treated bovine embryos. (a) Percentage of nuclei found in me&phase at several periods after removal of aphidicolin (closed circles) compared with controls (open circles), and (b) percentage of blastomems positively showing BrdU incorporation in tmated embryos several hr after removal of aphidicolin compared with controlsVertical bars indicate the standard error among replicates. Toxicity To assess the toxicity of aphidicolin on embryo development in vitro, 180 eight-cell control and treated embryos exposed for 12 h to concentrations of aphidicolin ranging from 0.7 to 2.8 pM were washed and cultured in aphidicolin-free MB2 medium for a further 6 d (Figure 4). Concentrations above 0.7 l.tM used in this study, including the minimal effective dose of 1.4 )IM, had a significant effect on the ability of treated embryos to develop to the blastocyst stage. At these concentrations, a significant effect was also observed on the number of cells per blastocyst when compared with the controls (P&.05). Further, the hatching rate of blastocysts at Day 6 of culture was also affected when using the minimal effective dose of 1.4 pM as compared with that of the controls (P&.05; Figure 4). DISCUSSION Our results indicate that cleavage to the fifth cell cycle can be successfully arrested in 8-c&l bovine embryos with aphidicolin at 0.7 l&I. Moreover, resumption of DNA synthesis does not occur in treated embryos up to 4 h after removal from aphidicolin as assessed by BrdU incorporation. These results are supported by previous findings where complete cleavage arrest with aphidicolin was obtained with bovine &cell embryos exposed at 50 h but only partially at 74 h post insemination (25). Aphidicolin has been shown to act by specifically inhibiting DNA polymerase alpha, preventing DNA synthesis at the S phase of the cell cycle and, thereby, arresting embryos in interphase (29). In this study, less than 1% of blastomeres were incorporating BrdU at the end of treatment and up to 2 h after removal of aphidicolin, indicating the powerful effect of this agent on DNA polymerase alpha inhibition. Incorporation of BrdU increased by 4 h (38%) to reach a maximum of 55% by 8 h before decreasing to 12% 10 hr after removal from aphidicolin.
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100 P, 80 A B 60 B -40 3 e 20 8: 0
l(a)
PcO.05 I a
a
0.70 control 1.40 Concentration of aphidicolin (PM)
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Figure 4 Effects of aphidicolin at doses of 0.7 to 2.8 pM on, (a) the in vitro development of g-cell bovine embryos to the blastocyst stage, (b) the hatching rate and, (c) the number of nuclei at Day 6after removal of the agent. Hatched bars indicate effects of aphidicolin at the minimal effective dose of 1.4 PM of aphidicolin. N=180 per experiment_ Different superscripts indicate 5% level of significance (P&05). Metaphase-like chromatin was observed in 43% of the blastomeres at the end of tmaunent, suggesting that aphidicolin may cause metaphase arrest in some g-cell stage blastometes. Although the mechanisms leading to the metaphase arrest observed in our studies am unknown, it is possible that aphidicolin induces irreversible chromatin condensation leading to cellular death. This assumption is supported by the stable percentage of metaphase nuclei present during several hours after release, suggesting that a percentage of nuclei are not re-entering the cell cycle. Further, lower cell numbers at the blastocyst stage may have resulted from this irreversible arrest of some blastomeres at the g-cell stage. Aphidicolin may affect the major transcriptional activation that occurs at the g-cell stage in cattle (3,16) and, in such way, lead to irreversible damage and blockage of cleavage to the fifth cell cycle. In mice, inhibition of DNA synthesis in Gl preceding transcriptional
Theriogenology
activation at the second cell cycle prevents later differentiation events regardless of the occurrence of a “catch-up” round of DNA synthesis immediately after the removal of the inhibitor (28). Observations on the inability of several treated embryos to develop to the blastocyst stage, a significant decrease in the number of cells per blastocyst and in the hatching rate, indicates that aphidicolin is detrimental when used for bovine S-cell stage blastomere synchronization. Toxicity to aphidicolin has been reported to be stage-dependent in mouse embryos, with toxic effects having been observed before (1,9,10,29) and after the 8-cell stage where aphidicolin treatment led to a deficiency in inner cell-mass (ICM) cell number in blastocysts (10,13,22,29). In the latter case, normal ICM development was obtained when cell number was increased by aggregating embryos during aphidicolin treatment, suggesting that this deficiency was due to the low cell number rather than the inability of ICM to form after exposure to the artest agent (10). In cattle, no data are available on stage-dependent developmental sensitivity to aphidicolin and it is yet unclear whether treatment of bovine embryos at other stages than 8-cell will be less detrimental to normal development, Although it is possible that, due to the activation of transcription, bovine 8-cell stage am particularly sensitive to exposure periods lasting 12 h, other cell cycle arrest-agents have shown little or no detrimental effects on development (23,24). Since the latter studies were able to induce a synchronous entry into the fifth cell cycle within 3 to 4 h after removal, it is possible that short-term exposures to aphidicolin following treatment with nocodazole or 6-Dimethylaminoprine would enable precise cell cycle arrest at the Gl to S-phase transition with fewer detrimental effects to development to the blastocyst stage. Similar approaches have been used with somatic cell lines using arrest at GO by serum starvation followed by the addition of serum in the presence of either aphidicolin (33) or mimosin (17) to arrest the cells at the GUS boundary. After removal from the DNA synthesis inhibitors, cells proceeded synchronously through the S and G2 phases, allowing manipulations at specific points in either phase. Further studies using multiple synchronization protocols are required to enable proper and reversible cell cycle arrest of cattle pteimplantation embryos. REFERENCES 1. Alexandre H. Effet de l’inhibition specitique de la replication de I’ADN par 1’Aphidicoline sur la differentiation primaim de l’oeuf de souris en preimplantation. Cons Rech Acad Sci Paris 1982; 294: 1001-1006. 2. Barnes FL, Collas P, Powell R, Ring WA, Westhusin M, Shepherd D. Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown. chromosome constitution, and development in nuclear transplant bovine embryos. Dev Biol 1993; 36: 33-41. 3. Camous S, Kopecny V, Flkhon J-E. Autoradiographic detection of the earliest stage of (3H)uridine incorporation into the cow embryo. Biol Cell 1986; 58: 195200. 4. Campbell KHS, Loi P, Cappai P, Wilmut I. Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol Reprod 1994; 50: 13851393. 5. Campbell RI-IS, Ritchie WA, Wilmut I. Nuclear-cytoplasmic interactions during the fust cell cycle of nuclear transfer reconstructed bovine embryos: Implications for deoxyribonucleic acid replication and development. Biol Reprod 1993; 49: 933-942. 6. Cheong I-IT, Takahashi Y, Kanagawa H. Birth of mice after transplantation of early cell cycle stage embryonic nuclei into enucleated oocytes. J Reprod Fertill993; 48: 958-963. 7. Collas P, Balk JJ, Rob1 JM. Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol Reprod 1992; 46: 492-500. 8. Collas P, Pinto-Correia C, DeLeon FAP, Rob1 JM. Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos. Biol Reprod 1992; 46: 501-511. 9. Cozad KM, Warner CM. The effects of aphidicolin and a-amanitin on DNA synthesis in preimplantation mouse embryos. Gamete Res 1982; 6: 155-160. 10. Dean WL, Rossant J. Effect of delaying DNA replication on blastocyst formation in the mouse. Differentiation 1984; 26: 134-137.
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11. Gag& M, Pothier F, Sirard M-A Effect of microinjection time during post-fertilization S-phase on bovine embryonic development Mol Reprod Dev 1995; 41: 184-194. 12. Hidaka H, Tanaka T. Naphthalenesulfonamides as cahnodulin inhibitors. Meth Enzym 1983; 102: 185194. 13. Howlett SK The effect of inhibiting DNA replication in the one-cell mouse embryo. Roux’s Arch Dev Biol1986; 195: 499-505. 14. Hutchmson CJ, Brill D, Cox R, Gilbert J, Kill I, Ford CC. DNA replication and cell cycle control in Xenopus egg extracts. J Cell Sci 1989; 12: 197-212. 15. Kelly SJ, Mulnard JG, Graham CF. Cell division and cell allocation in early mouse development. J Embryo1 Exp Morph01 1978; 48: 37-51. 16. Kopecny V, Flechon JE, Camous S, Fulka J,Jr.. Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine structural autoradiographic study. Mol Reprod Dev 1989; 1: 79-90. 17. Grren DK, Petersen LN, Bohr VA. A W-responsive G2 checkpoint in rodent cells. Mol Cell Biol 1995; 15: 3722-3730. 18. Gtaegui PJ, G’Neil GT, Campbell KHS, Wilmut I. Transfer of nuclei from 8-cell stage mouse embryos following use of Nocodazole to control the cell cycle. Mol Reprod Dev 1994; 39: 147152. 19. Ouhibi N, Fulka J,Jr., Kanka J, Moor RM. A reversible block at the GUS border during cell cycle progression of mouse embryos. Int J Dev Biol 1994; 38: 731-736. 20. Parrish JJ, Susko-Parrish JL, Leibfriedge-Ruthedge ML, Critser ES, Eyestone WH, First NL. Bovine in-vitro fertilization with frozen thawed semen. Theriogenology 1986; 25: 591-600. 21. Poueymirou WT, Schultz RM. Regulation of mouse pmirnplantation development: inhibitory effect of cahnodulin antagonist W-7 on the first cleavage. Mol Reprod Dev 1990; 26: 2 11-216. 22. Samake S, Smith LC. Effect of cell-cycle-arrest agents on cleavage and development of mouse embryos. J Exp Zoo1 1995; 274: 11 l-120. 23. Samake S, Smith LC. Synchronization of cell division in bovine embryos produced in vitro: effects of nocodazole. Mol Reprod Dev 1996; 44: 486-492. 24. &make S, Smith LC. Synchronization of cell division in eight-cell bovine embryos produced in vitro:effectsof 6-dimethyllaminopurine. J Reprod Fertil1997; 110: 21-27. 25. Smith LC, Meimlles FV, Bustin M, Clarke HJ. Assembly of somatic histone Hl onto chromatin during bovine early embryogenesis. J Exp Zoo1 1995; 273: 317-326. 26. Smith LC, Wilmut I, Hunter RHF. Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos. J Reprod Fertil 1988; 84: 619-624. 27. Smith LC. Wihnut I, West JD. Control of fast cleavage in single-cell reconstituted mouse embryos. J Reprod Fertil 1990; 88: 655-663. 28. Smith RKW, Johnson MH. DNA replication and compaction in the cleaving embryo of the mouse. J Embryo1 Exp Morph01 1985; 89: 133-148. 29. Spindle A, Nagano H, Pedersen RA. Inhibition of DNA replication in pmimplantation mouse embryos by aphidicolin. J Exp Zoo1 1985; 235: 289-295. 30. Stice SL, Keefer CL, Matthews L. Bovine nuclear transfer embryos: oocyte activation prior to blastomete fusion. Mol Reprod Dev 1994; 38: 61-68. 31. Surani MAH, Barton SC. Spatial distribution of blastomeres is dependent on cell division order and interactions in mouse mot&e. Dev Biol1984; 102: 335343. 32. Sutherland AE, Speed TP, Calarco PG. Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage. Dev Biol 1990, 137: 13-25. 33. Tobey RA, Valdez JG, Crissman HA. Synchronization of human diploid tibroblasts at multiple stages of the cell cycle. Exp Cell Res 1988; 179: 400-416. 34. Valdimarsson G, Kidder GM. Temporal control of gap junction assembly in preimplantation mouse embryos. J Cell Sci 1995; 108: 1715-1722. 35. Wormd DM, Turner BM, Schultz RM. Temporally mstricted spatial localization of acetylated isoforms of hitone H4 and RNA polymerasc II in the 2-tell mouse embryo. Development 1995; 121: 2949-2959.