Voi 5 (1996): 529-634. A mouse model for improving cell survival of bisected cattle embryos Peter Bredbacka 1 Agricultural Research Centre ofFinland, Institute ofAnimal Production. Finland Morula and blastocyst stage embryos recovered from B6D2F! mice were bisected with a metal micro- blade in M 2 medium with or without sucrose and/or cytochalasin B supplementation. Cell lysis was determined by staining the embryos with Hoechst 33258 and propidium iodide. Lysed cells take up both stains but non-lysed cells only the Hoechst 33258 stain, resulting in pink fluorescence for lysed cells and blue fluorescence for non-lysed cells under UV excitation. During bisection of morulae, the presence of cytochalasin B decreased the proportion of lysed cells in both the absence (P=0.000l) and presence of sucrose (P=0.001). During bisection of blastocysts the average proportion of lysed cells was slightly lower in the presence of cytochalasin than that in the control medium, but the effect was not statistically significant (P=0.34). No effect of sucrose was observed in either demi-morulae or demi-blastocysts. These results are essentially similar to those obtained in simultaneous experi- ments with cattle embryos, suggesting that the simpler mouse model might be useful for developing less traumatic bisection protocols for cattle embryos. Key words: demi-embryo, splitting, cell lysis, propidium iodide, Hoechst 33258, cytochalasin, sucrose 'Present address: Finnish Animal Breeding Association, P.O. Box 40, FIN-01301 Vantaa, Finland, e-mail: peter.bredbacka@mloy.fi Introduction Embryo bisection can be used in embryo trans- fer programmes either to increase the number of transferable (demi)embryos or to produce iden- tical twin calves for research or other purposes. The first successful transfers of bisected bovine embryos were reported in the early 1980 s (Wil- ladsen et al. 1981, Ozil et al. 1982). Although embryo bisection yields more off- spring per original embryo under appropriate con- ditions, subsequent splitting of an embryo does not seem to give any further benefit, as the viability of quarter embryos is roughly half that of demi-em- bryos (Willadsen et al. 1981, Bredbacka et al. 1992). Demi-embryos apparently are particularly sensitive to further invasive treatments. Substan- tial cell loss in bisected embryos will either com- promise further development before implantation © Agricultural and Food Science in Finland Manuscript received September 1996 529 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=SL2vJNhsLuei98GX.UkGkxXz-AR-oc0paYpf66Q.rBC2iJeLY7VK4m5AZRMqH919INiUguEl76SRZEWdqM8q3IPJg8JIsGmcURsgSn9FD5oPkhPMyq2mNyAeZEcXukP1BDIoLmEfWTKX_9_MZIPfgGY-A62A8qUYM-MyXS1fO71Sv7i0vw6815i0dFlAeEUhPycrYhPqy5DcZXTV2cGcXfIEkAcY17dey2eYNq1pcTRE5x_6KWNmlnaEylHCfSd86FnkMB_eXBYhf7M5owuA6yd5v-HlPux3vXo--gBvleVABvcA Bredbacka, P. Cell lysis in demi-emhryos or result in an insufficient embryonic signal for maternal recognition, or both. The success of producing live foetuses after splitting varies, depending upon factors such as bisection technique (Merles and Bondioli 1985), embryo age and stage (Williams et al. 1984, McEvoy and Sreenan 1990), method of transfer (Takeda et al. 1986), and type and status of re- cipient (Arave et al. 1987, Bredbacka et al. 1992). However, data on the survival of cells during bisection are sparse, as are the published data on attempts to increase viability of blast- omeres during the splitting process. A recent report (Bredbacka 1995) demonstrates that a considerable proportion (about 20% to 30%) of blastomeres lyses in embryos bisected using a microblade-assisted technique. The effectiveness of the technique is nevertheless confirmed by a 60% pregnancy rate ofdemi-embryos transferred singly (Bredbacka et al. 1996). Survival after serial splitting might be significantly improved, if cell lysis could be prevented more efficiently. The major factor holding up improvements to bovine embryo bisection protocols is the cost of embryos. Although in vitro produced (IVP) em- bryos represent a relatively inexpensive alterna- tive to in vivo produced embryos, the IVP technol- ogy is more elaborateand requires embryo culture facilities. Furthermore, regardless of the method of embryo production, bovine embryos exhibit a large variation in morphology, which sometimes complicates interpretation of results. The aim of my study was to demonstrate the feasibility of a mouse model for predicting cell survival in bovine embryos. The effect of sucrose and cytochalasin B on cell survival was investi- gated with differential staining of lysed and non- lysed cells. The results were compared with those of a parallel study in the bovine (Bredbacka 1995). Material and methods Female B6D2FJ (C578L6 x DBA Fj) mice were first superovulated by injecting 5 to 7.5 IU PMSG and 5 IU hCG 48 h apart and then mated with B6D2F, males. Compacted morulae were recovered 73 to 75 hours after the hCG injec- tion and blastocysts were recovered 90 to 94 h after the hCG injection using the method devised by Hogan et al. (1986). The nuclei of blastomeres were stained by exposing the embryos to 20 pg/ml Hoechst 33258 stain in 4-well Nunc dishes (Nunc, Cat No. 176740, Roskilde, Denmark) for 3 to 5 h at 37%C in air. The stain was diluted in 500 pi of M 2 medium (Quinn et al. 1982) containing 4 mg/ml bovine serum albumin (M2+BSA). The M 2 was made up of analysis grade chemicals and the BSA was from Sigma (Cat. No. A-9647, St. Lou- is, MO, USA). Each embryo was evaluated im- mediately before splitting using a Leitz Fluovert inverted microscope; embryos with any indica- tion of abnormal morphology were excluded from the experiments. The control medium for bisection was M2+BSA. In the treatment groups the sucrose was added at a concentration of 200 mM, and cytochalasin B (Sigma, Cat. No. C6762, St. Lou- is, MO, USA) was used at 7.5 (Jg/ml. The com- bination of cytochalasin B and sucrose was also tested in the experiments with morulae. Before being split in sucrose-containing medium, the embryos were allowed to equilibrate for 3 to 5 min in the medium at room temperature. When bisected in the presence of cytochalasin B, the embryos had an equilibration time of sto 15 min at room temperature. The embryos were bisected with a Leitz mi- cromanipulator using a microblade prepared from a razor blade (Williams and Moore 1988) and the “scratched bottom technique” (Bred- backa 1991). With this technique, scratches are produced parallel to the orientation of the mi- croblade on the bottom of the lid of a plastic petri dish (Nunc, Cat. No. 153066, Roskilde, Den- mark) to prevent the embryo from slipping dur- ing bisection. The embryo is then bisected by a vertical movement of the microblade in a drop of 100-200 pi of medium. Blastocysts are bi- sected so that both the trophectoderm and inner cell mass are halved. To reduce the stickiness of 530 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 529-534. blastomeres the stage was chilled to about 10- 15°C by placing precooled aluminiumblocks on the Leitz Fluovert microscope stage. The tem- perature of the stage was monitored by a Linkam CO6O stage warmer with the heating off. After the splitting, each demi-embryo pair was placed in a 50 pl-drop of M2+BSA. Propid- ium iodide (Sigma, Cat. No. P-4170, St. Louis, MO, USA) was added to a final concentration of 10 pg/ml to stain nuclei of lysed cells. After 45 to 60 min of staining at room temperature, each demi-embryo pair was washed and placed into 50 pi of medium in a well of a 96-well plas- tic dish. The nuclei were counted by fluorescence microscopy using a Leitz Fluovert microscope with filter block A. Cells of each embryo were classified as lysed (pink fluorescence) or viable (blue fluorescence), with the results being expressed as the proportion of the total number of cells. Data were analysed by variance analysis using the Statistical Analysis System (SAS Institute, Cary, NC, USA). Results The average number of nuclei (±SEM) in moru- lae was 18.5 (±0.6) and in blastocysts 47.3 Table 1. Cell viability of mouse morulae after splitting in different media. Treatment n Proportion (±SEM) P value" of viable cells Control 24 0.41 (±0.03) Cytochalasin B 24 0.56 (±0.02) 0.0001 Sucrose 24 0.43 (±0.02) 0.52 Cytochalasin B + sucrose 24 0.55 (±0.03) 0.0001 1 In comparison with control medium Table 2. Cell viability of mouse blastocysts after splitting in the presence of cytochalasin B, Treatment n Proportion (±SEM) P value* of viable cells Control 19 0.47 (±0.02) Cytochalasin B 20 0.49 (±0.02) 0.34 * In comparison with control medium Table 3. Cell viability of mouse blastocysts after splitting in the presence ofsucrose. Treatment n Proportion (±SEM) P value* of viable cells Control Sucrose 20 0.45 (±0.02) 20 0.45 (±0,02) 0.96 * In comparison with control medium. Fig. I. Brightfield and fluorescence image of a mouse demi-morula. Pink fluorescence indicates stain- ing of nuclei of lysed cells and blue fluorescence staining ofnuclei with intact cells. (Photos: Peter Bred- backa). 531 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka, P. Cell lysis in demi-embryos (±0.9). After the staining, the nuclei of most blas- tomeres along the plane of the bisection embry- os typically displayed pink fluorescence as an indication of propidium iodide uptake, whereas the remaining nuclei showed blue fluorescence (see Fig. 1). The results of the bisection of moru- lae are summarized in Table 1. When compared with bisection in control medium, an increase in the proportion of viable (non-lysed) cells was found in media supplemented with cytochalasin B, whereas no such effect was found with the addition of sucrose. Cytochalasin also decreased cell lysis in the presence of sucrose (P=0.001). Table 2 summarizes the results of bisecting blastocysts in medium containing cytochalasin B. The effect of the addition of sucrose on cell survival during bisection of blastocysts is sum- marized in Table 3. No beneficial effects of the treatments on cell viability in blastocysts were observed. Discussion Using the same method as in this study a previ- ous investigation demonstrated thatabout a quar- ter of the cells in Day 7 cattle embryos (about 20% in morulae, 25-30% in blastocysts) lyse when bisected with a metal blade (Bredbacka 1995). The present study suggests that the pro- portion of lysed cells in bisected mouse embry- os is even higher than in cattle embryos. About half of the blastomeres expressed membranerup- ture as indicated by uptake ofpropidium iodide. One possible explanation for this difference is the lower cell numbers in mouse morulae and blastocysts: in the previous experiment (Bred- backa 1995) cattle morulae had 2-4 times as many nuclei and cattle blastocysts about twice as many nuclei as the corresponding develop- mental stages of mouse embryos in this study. Consequently, in the mouse, a higher proportion of cells would be located in the plane of bisec- tion. It is also possible that mouse blastomeres are more sensitive to damage from bisection. Cytochalasins inhibit reversibly the polym- erization of actin, thus enhancing the flexibility of cell membranes. This property has been used to decrease cell damage in nucleus transfer ex- periments (for review, see Loskutoff 1990). Cy- tochalasin B has been shown to decrease cell lysis during bisection of Day 7 bovine embryos (Bredbacka 1995). In the present study, treatment of mouse morulae with cytochalasin B signifi- cantly decreased the proportion of lysed cells in bisected morulae. Note that a highly significant difference (P=0.0001) from the control medium could be shown with only a small number of embryos (24 per treatment). Despite the clearly beneficial effect of cyto- chalasin B in morulae, no clear effect of this compound was demonstrated in blastocysts. Since the proportion of non-lysed cells was slightly higher in the presence of cytochalasin, it is possible that the number of embryos was too low to demonstrate an actual effect. On the other hand, cytochalasin may provide less pro- tection for cells in blastocysts. It is also possi- ble that the inner cell mass (ICM) and the troph- ectoderm (TE) respond differently to cytochala- sin. If cytochalasin protects only one of the two cell types, variation in the ICM/TEratio between embryos would mask the actual effects. This stresses the need to use more embryos for anal- ysis unless cell survival can be measured sepa- rately in the ICM and TE. It has been proposed, that adding of sucrose to the splitting medium inhibits pressure-induced cell membranerupture during the splitting proc- ess by means of dehydration (Herr et al. 1988). In the present study the addition of sucrose at a concentration of 200 mM had no beneficial ef- fect on mouse blastomere survival. The same conclusion was drawn in the study with bovine embryos (Bredbacka 1995). Addition of sucrose at another concentration, however, might have rendered the blastomeres more resistant to the stress induced by the bisection procedure. The observations made here with mouse morulae are essentially identical to the results obtained with cattle embryos (Bredbacka 1995), suggesting that mouse embryos might be useful 532 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 529-534. for developing splitting techniques in cattle. In an experiment with a limited number of equine embryos, cytochalasin B tended to protect cell lysis (Huhtinen et al. 1995), implying the possi- bility of using the mouse model across several species. Bringing cell lysis down to a minimumcould improve the efficiency of embryo transfer pro- grammesnot only by making bisection less trau- matic, but also by improving biopsy for preim- plantation diagnosis. Increased cell viability of biopsied embryos could have a substantial ef- fect on pregnancy rates when biopsy is combined with cryopreservation. The use of mouse embryo bisection as a mod- el for improving the cattle embryo splitting pro- tocol has several advantages. The cost of pro- ducing mouse embryos is reasonable especially compared with in vivo produced cattle embryos. Although the use of the latter need not be ruled out, mouse embryos show less variability in qual- ity as assessed by morphological criteria, not least because inbred lines or hybrid F, lines can be used. The dead cells often present in intact cattle embryos complicate splitting experiments, as lysed cells take up propidium iodide even without the cell injury induced by microsurgery and so have to be excluded from analysis. Last, but not least, with about half of the cells lysing in a standard splitting protocol with mouse em- bryos, improvements to the protocol are easier to measure than with cattle embryos, in which roughly 25% of the cells lyse in the standard protocol. In conclusion, the use of mouse embryos of- fers a valuable tool for selecting protocols for further bisection and biopsy studies with cattle embryos. Acknowledgements. I thank T.-M. Nieminen for manage- ment of the mice and preparation of the media used in this study and K. Bredbacka for useful suggestions with regard to the manuscript. References Arave, C.W., Bunch, T.D., Mickelsen, C.H. & Warnick, K. 1987. Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd. Theriogenology 28: 373-382. Bredbacka, P. 1991. Biopsy of morulae and blastocysts. Reproduction in Domestic Animals 26: 82-84. - 1995. Factors affecting cell viability during bisection of bovine embryos. Theriogenology 44: 159-166. -, Huhtinen, M., Aalto, J. & Rainio, V. 1992. Viability of bovine demi- and quarter-embryos after transfer. Theri- ogenology 38: 107-113. -, Jaakma, 0. & Miiursepp, 1. 1996. Production of calves following nonsurgical transfer of fresh and refrigerated bovine demi-embryos. Agricultural and Food Science in Finland 5: 521-527. Herr, C., Holt, N. & Reed, K.C. 1988. Effect of sucrose and calcium in the splitting medium on survival of quartered ovine morulae. Proceedings of the Annual Conference of the Aus- tralian Society of Reproductive Biology. 20:10. (Abstract). Hogan, 8., Constantini, F. & Lacy, E. 1986. Manipulat- ing the Mouse Embryo: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, p. 92-95. Huhtinen, M., Bredbacka, P. & Kotilainen,!. 1995. Non- surgical transfer of DAPI-stained equine demi-embryos treated with cytochalasin B and nocodazole. Biology of Reproduction, Monograph Series No, 1: 325-328. Loskutoff, N.M. 1990. Micromanipulation of embryos and gametes. Embryo Transfer Newsletter 8: 5-14. McEvoy, T.G. & Sreenan, J.M. 1990. Effect of embryo quality and stage of development on the survival of zona pellucida-free cattle demi-embryos. Theriogenology 33: 1245-1253. Merles, P.C. & Bondioli, K.R. 1985. Effect of splitting technique on pregnancy rate from half embryos. Theri- ogenology 23: 209, (Abstract). Ozil, J.P., Heyman, Y. & Renard J.P. 1982. Production of monozygotic twins by micromanipulation and cervical transfer in the cow. Veterinary Record 110: 126-127. Quinn, P., Barros, C. & Whittingham, D.G. 1982. Pres- ervation of hamster oocytes to assay the fertilizating ca- pacity of human sperm. Journal of Reproduction and Fertility 66: 161-168. Takeda, T., Hallowed, S.V., McCauley, A.D. & Hasler, J.F. 1986. Pregnancy rates with intact and split embryos transferred surgically and nonsurgically. Theriogenology 25: 204. (Abstract). Willadsen, S.M., Lehn-Jensen, H., Fehilly, C.B. & New- comb, R. 1981. The production of monozygotic twins of preselected parentage by micromanipulation of non-sur- gically collected cow embryos. Theriogenology 15: 23- 29, Williams, T.J., Elsden, R.P. & Seidel, G.E., Jr. 1984. Pregnancy rates with bisected bovine embryos. Therio- genology 22: 521-531. - & Moore, L. 1988. Quick-splitting of bovine embryos. Theriogenology 29: 477-484. 533 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Bredbacka , P. Cell lysis in demi-embryos SELOSTUS Hiiren alkio mallina solukestävyyden parantamiseksi naudan alkioiden halkaisussa Peter Bredbacka Maatalouden tutkimuskeskus Morula- ja blastokystivaiheessa olevia hiiren alkioi- ta halkaistiin mikroterällä M2-liuoksessa. Halkaisu- kokeessa tutkittiin sakkaroosin (200 mM) ja sytoka- lasiinin (7,5 ug/ml) vaikutusta solujen kestävyyteen kaksoisvärjäyksen avulla. Värjäyksen jälkeen solut voidaan erottaa toisistaan fluoresenssimikroskoopin avulla värin perusteella, Hoechst 33258-väri värjää kaikki solut (sininen fluoresenssi), ja propidiurnjodi vain tuhoutuneet solut (vaaleanpunainen fluoresens- si). Morulavaiheessa olevia alkioita halkaistaessa sy- tokalasiini vähensi hajonneiden solujen osuutta riip- pumatta siitä, oliko liuoksessa myös sakkaroosia. Kun käytettiin blastokystivaiheessa olevia alkioita, eri käsittelyillä ei havaittu vaikutusta hajonneiden solu- jen määriin. Sakkaroosilla ei ollut vaikutusta blasto- kystivaiheen solujen kestävyyteen. Nämä tulokset ovat samansuuntaisia kuin vastaavasta naudan al- kioilla tehdystä kokeesta saadut tulokset. Halkaisussa tuhoutuneiden solujen erottaminen on hiiren alkiolla helpompaa kuin naudan alkioilla. Edullisena ja yk- sinkertaisena hiirimalli voi olla hyödyllinen kehitet- täessä naudan alkion soluja paremmin suojaavia mik- rokirurgisia menetelmiä. 534 AGRICULTURAL AND FOOD SCIENCE IN FINLAND