BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ 5 DOI 10 15625/vap 2022 0095 THE EFFECT OF HISTONE DEACETYLATION INHIBITORS (HDACI) TREATMENT DURING[.]
BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM - HỘI NGHỊ KHOA HỌC QUỐC GIA LẦN THỨ DOI: 10.15625/vap.2022.0095 THE EFFECT OF HISTONE DEACETYLATION INHIBITORS (HDACI) TREATMENT DURING ZYGOTIC GENE ACTIVATION ON PREIMPLANTATION DEVELOPMENT OF CLONED BOVINE EMBRYOS Do Minh Tan1,#, Nguyen Mai Phuong1,#, Cao Hoang Nam1, Nguyen Tuan Anh1, Nguyen Huu Hoang Minh1, Pham Minh Chien1, Pham Quoc Dinh1, Nguyen Van Thuan1,* Abstract: The born of Dolly sheep marked the success of cloned animals by SCNT technique Until now, many healthy cloned animals were born such as mouse, cow, and monkey Moreover, cloning technique also carried potential future applications such as animal preservation and human therapy Therefore, a lot of research focus into improve the success of producing cloned animals This study aimed to examine the effect of HDACi treatment during the ZGA period In this study, the effect of HDACi treatment with different concentrations on preimplantation development of cloned bovine embryos is done SCNT bovine oocytes treated with TSA nM + SCR 125 nM reached the highest rate at morula stage (35.0 %) while untreated group and TSA 10nM + SCR 250 nM groups was only 21.7 % and 21.1 %, respectively Continually, the rate of cloned bovine blastocyst was 20.0 % for TSA nM + SCR 125 nM The second experiment was optimizing timing treatment during ZGA for the second step of cloned bovine embryos According to the result, HDACi treatment at the first step after activation and the second step at 52–62 h after activation had 28.6 % of cloned embryos developed to the blastocyst stage In conclusion, 2-steps treatment of TSA nM + SCR 125 nM in which the first step occurs during embryo activation timing prolonged to 10 hours and the second step at 52 hours post activation prolonged to 62 hours post activation improved the preimplantation development of cloned bovine embryos and cloned blastocyst formation rates Keywords: Bovine cloning, HDACi, SCNT, zygotic gene activation INTRODUCTION The first success of somatic cell nuclear transfer (SCNT) technique on mammalian was labeled by the born of Dolly sheep (Wilmut et al., 1997) However, for more than twenty-years so far, the success rate of producing cloned offspring remained extremely low, only about 2-10 % (Keefer, 2015) Many pieces of evidence demonstrated that abnormal epigenetic modification involves in DNA methylation and histone modification such as acetylation, methylation, phosphorylation and ubiquitylation are likely associated International University, Vietnam National University, Ho Chi Minh City Equally contributed to this work * Email: nvthuan@hcmiu.edu.vn # 850 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM with failure in the reprogramming process This led to the low success of producing healthy cloning animals (Kishigami et al., 2006) Therefore, a series of studies have tried to prevent the epigenetic errors which are expected to enhance the animal cloning efficiency Several strategies showed that the application of histone deacetylation inhibitors (HDACi) has been reported to enhance histone acetylation, nuclear reprogramming, gene expression and full-term development (Thuan et al., 2009; Liu et al., 2018) Among these approaches, Trichostatin A (TSA) and Scriptaid (SCR), two members of HDACs was used to induce transcriptional activity, decrease DNA methylation, enhance the preimplantation development of bovine SCNT embryos (Lee et al., 2010) In addition, the bovine cloning efficiency could be improved by SCR (Wang et al., 2011) with lower cellular toxicity impact than TSA However, the rate of success in producing cloned offspring was still low evenly after single treatment of HDACi at the first ten hours after artificial activation In 2013, a research demonstrated the positive effects of combining TSA and SCR on rabbit cloning (Chen et al., 2013) Besides, the zygotic gene activation (ZGA) was well proved as major event associated the development of bovine embryos and it occurred at the 8/16 cell stage (Graf et al., 2014) Those were reasons leading to study the effect of HDACi treatment during the ZGA of cloned bovine embryos Thus, the purpose of this study was to evaluate and optimize the two-steps HDACi treatment for improving the developmental competence of clone bovine embryos, which directly related to the success of bovine cloning MATERIALS AND METHOD 2.1 In-vitro maturation (IVM) of bovine oocytes The cumulus-oocyte complexes (COCs) were aspirated from the 4-6 mm follicles from bovine ovaries About 10-15 good quality COCs were cultured in the 150 L floating drops of IVM medium including TCM 199 medium supplement with 10 % fetal bovine serum (FBS), 50 g/mL sodium pyruvate (Napy), 50 ng/mL epidermal growth factor (EGF), 0065 IU/mL luteinizing hormone (LH), mg/mL follicle stimulating hormone (FSH), and g/mL estradiol (ES) covered by mineral oil and cultured at 38.5 oC and % CO2 in a humidified atmosphere After 18-20 hours in-vitro maturation, the expanded cumulus cells were completely removed from oocytes by treatment with 0.1 % hyaluronidase in HEPES medium The mature bovine oocytes with first polar body (1st PB), homogenous cytoplasm and round shape were prepared for SCNT steps 2.2 Bovine fibroblast cell culture form bovine lung tissue Bovine lung tissues were obtained from the slaughterhouses and cut into small pieces for culture Bovine lung tissues were cultured with Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented Streptomycin mg/mL, Penicillin mg/mL and 10 % FBS in % CO2 and 38.5 oC in a humidified atmosphere After reaching around 80 % confluence of the culture dish, cells were passed and freezed at -196 oC for longterm use The cell for SCNT was used from passage 3rd to passage 7th PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 851 2.3 The enucleation of bovine oocyte Good quality matured bovine oocytes were loaded into HEPES medium with cytochalasin B (CB) drops for enucleation step The location of MII was labeled by XYClone laser (Hamilton Thorne) when the extruded MII was observed by inverted microscope (Nikon Eclipse Ti) When all of oocytes were labeled, the chromosome would be gently aspirated through enucleation micropipette (10-12 µm inner diameter – ID) 2.4 The injection method after nucleation method The bovine fibroblast cells from the 5th to 7th passage were used for injection The cells at G0/G1 phase were used for SCNT by observing morphology and surface of cells Cell membranes were removed by using injection pipettes with 7-8 µm The single nucleus from the donor cell was transferred into enucleated bovine oocytes by microinjection using Piezo-actuated micromanipulation (PMAS-CT150) 2.5 Chemical activation and in vitro development of cloned bovine embryos For activation, the reconstructed SCNT bovine oocytes were activated by µM ionomycin for minutes and mM 6-Dimethylaminopurine for hours at 38.5 oC and % CO2 humidified atmosphere in modified synthetic oviduct fluid (mSOF) medium supplemented with mg/ml BSA Embryos were immediately treated with different concentration of SCR and TSA after being activated for 10 hours following to treatment at 52-62 hours post activation (hPA) The quality and the development of cloned bovine embryos were recorded and analyzed at 2-cell stage, 4-cell stage, 8-cell stage, morula stage and blastocyst stage 2.6 Data analysis Data were analyzed by One-way ANOVA with Least-Significant Difference (LSD) test using IBM SPSS Statistics 20 software The probability P < 0.05 was considered as significant difference All the experiments were replicated at least times RESULT AND DISCUSSION 3.1 Effect of Trichostatin A and Scriptaid treatment on preimplantation development of cloned bovine embryos According to Table 1, the result showed that from the two-cell stage to the eight-cell stage embryos, there was no significant difference between the experimental groups However, at morula stage, there were significant differences between combined TSA and SCR treatment at different concentrations Only the group of cloned embryos treated with TSA 5nM + SCR 125 nM reached highest percentage (35.0 %) at morula stage while untreated group and TSA 10 nM + SCR 250 nM group were only 21.7 % and 21.1 %, respectively Although the efficiency of TSA was demonstrated in the preimplantation of cloned embryos by many researchers, the full-term development of cloned embryos was limited (Meng et al., 2009) TSA treatment may cause toxicity to the post-implantation of cloned bovine embryos at high concentration Moreover, SCR 250 nM treatment was demonstrated with positive effect on the development of cloned bovine embryos BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 852 development based on the previous studies Therefore, in combination treatment of TSA and SCR, the concentration of TSA should be reduced to nM in combination with 125 nM SCR in order to limit the negative effect on the preimplantation and post-implantation of cloned bovine embryos Table Effect of Combine Trichostatin A and Scriptaid A treatment concentration on preimplantation development of cloned bovine embryos Group Control TSA nM + SCR 125 nM TSA 10 nM + SCR 250 nM Number (%) of cloned bovine embryos develop to 2-cell 4-cell 8-cell Morula Blastocy n (%) (%) (%) (%) st (%) 18 14 11 23 (78.3) (60.9) (47.8) (21.7)a (13.0)a 17 15 11 20 b (80.0) (75.0) (55.0) (35.0) (20.0)b 14 11 19 (73.7) (57.9) (42.1) (21.1)a (10.5)a In the same column, data with different letters are significantly different (P < 0.05) Each group was replicated three times 3.2 Effect of Trichostatin A and Scriptaid treatment timing on preimplantation development of cloned bovine embryos Figure The developmental rate of cloned bovine embryos at day The blastocyst stage of control group (A), TSA + SCR 1-step treatment, 2- step treatment with 2th treatment at 52 h and prolong 10 h (C), combine TSA + SCR 1-step treatment, 2- step treatment with 2th treatment at 62 h and prolong 10 h (D), 2- step treatment with 2th treatment at 62 h and prolong 20 h (E) Scale bar: 100 µm In the next experiment, the preimplantation development of cloned bovine embryos was evaluated from 2-cell stage to blastocyst stage to determine the treatment timing at the second step According to Table 2, there were significant differences from 2-cell to 8-cell stage between variety of experiment groups The rate of cloned bovine embryos at morula PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 853 stage was 39.3 % in the 2-steps treatment group of TSA 5nM + SCR 125 nM and reduced to 28.6 % at blastocyst stage While the rate of blastocyst was only 9.5 % for control group, 19.0 % for one-step treatment and 5.3 % for combined two-step treatment for 62-72 hPA at second step and no embryo could develop to blastocyst stage for the group that prolonged treatment duration to 20 hours for second step According to Wee et al., 2006, the level of AcH4K5 need to reach the highest level at 8-cell stage for the IVF bovine embryos and decreased from late 8-cell stage to blastocyst stage That leads to an explanation that the rate of cloned bovine embryos development down at morula stage and blastocyst with prolonging culture of cloned embryos with HDACi for 20 hours after 52 hPA Table Effect of Trichostatin A and Scriptaid A treatment during zygotic gene activation on preimplantation development of cloned bovine embryos Group Control TSA nM + SCR 125 nM (0-10 h) TSA nM + SCR 125 nM (0-10 h) – (52-62 h) TSA nM + SCR 125 nM (0-10 h) – (62-72 h) TSA nM + SCR 125 nM (0-10 h) – (52-72 h) n 21 21 Number (%) of cloned bovine embryos develop to 2-cell 4-cell 8-cell Morula Blastocy (%) (%) (%) (%) st (%) 16 12 10 b (76.1) (57.1) (47.6) (19.0) (9.5)a 17 16 13 c (80.9) (76.2) (61.9) (33.3) (19.0)b 28 23 (82.1) 22 (78.6) 17 (60.7) 11 (39.3)c (28.6)c 19 15 (78.9) 14 (73.7) 12 (63.2) (15.8)b (5.3)b 18 14 (77.8) 13 (72.2) (44.4) (5.6)a In the same column, data with different letter are significantly different (P < 0.05) Each group was replicated three times CONCLUSION In conclusion, combined treatment of TSA nM + SCR 125 nM could improve the success rate of cloned bovine embryos developed to the blastocysts stage Moreover, for 2-steps HDACi treatment, TSA nM + SCR 125 nM with first step during activation timing prolonged 10 h and the second step from 52-62 hPA improved the formation rate of cloned bovine blastocysts Acknowledgement: This study is funded by the Ministry of Science and Technology of Vietnam under grant number ĐTĐL.CN-49/16 854 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM REFERENCES Chen, C H., Du, F., Xu, J., Chang, W F., Liu, C C., Su, H Y., & Sung, L Y., 2013 Synergistic effect of trichostatin A and scriptaid on the development of cloned rabbit embryos Theriogenology, 79(9): 1284-1293 Graf, A., Krebs, S., Zakhartchenko, V., Schwalb, B., Blum, H., & Wolf, E., 2014 Fine mapping of genome activation in bovine embryos by RNA sequencing Proceedings of the National Academy of Sciences, 111(11): 4139-4144 Keefer, C L., 2015 Artificial cloning of domestic animals Proceedings of the National Academy of Sciences, 112(29): 8874-8878 Kishigami, S., Wakayama, S., Van Thuan, N., Ohta, H., Mizutani, E., Hikichi, T., & Wakayama, T., 2006 Production of cloned mice by somatic cellnuclear transfer Nature protocols, 1(1): 125-138 Lee, M J., Kim, S W., Lee, H G., Im, G S., Yang, B C., Kim, N H., & Kim, D H., 2010 Trichostatin A Promotes the Development of Bovine Somatic Cell Nuclear Transfer Embryos The Journal of Reproduction and Development, 57: 34-42 Liu, Z., Cai, Y., Wang, Y., Nie, Y., Zhang, C., Xu, Y., & Sun, Q., 2018 Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer Cell, 172: 881-887 Meng, Q., Polgar, Z., Liu, J., & Dinnyes, A., 2009 Live birth of somatic cell-cloned rabbits following trichostatin A treatment and cotransfer of parthenogenetic embryos Cloning and Stem Cells, 11: 203-208 Van Thuan, N., Bui, H T., Kim, J H., Hikichi, T., Wakayama, S., Kishigami, S., & Wakayama, T., 2009 The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice Reproduction, 138(2): 309 Wang, L J., Zhang, H., Wang, Y S., Xu, W B., Xiong, X R., Li, Y Y., & Zhang, Y., 2011 Scriptaid improves in vitro development and nuclear reprogramming of somatic cell nuclear transfer bovine embryos Cellular Reprogramming (Formerly" Cloning and Stem Cells"), 13(5): 431-439 Wee, G., Koo, D B., Song, B S., Kim, J S., Kang, M J., Moon, S J., & Han, Y M., 2006 Inheritable histone H4 acetylation of somatic chromatins in cloned embryos Journal of Biological Chemistry, 281: 6048-6057 Wilmut, I., A E Schnieke, J McWhir, A J Kind, and K H Campbell., 1997 Viable Offspring Derived from Fetal and Adult Mammalian Cells Nature, 385(8):10-13 PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 855 ẢNH HƯỞNG CỦA CHẤT ỨC CHẾ HISTONE DEACETYLATION TRONG GIAI ĐOẠN KÍCH HOẠT GEN HỢP TỬ (ZGA) LÊN KHẢ NĂNG PHÁT TRIỂN GIAI ĐOẠN TIỀN LÀM TỔ CỦA PHƠI BỊ NHÂN BẢN VƠ TÍNH Đỗ Minh Tấn1,#, Nguyễn Mai Phương1,#, Cao Hoàng Nam1, Nguyễn Tuấn Anh1, Nguyễn Hữu Hoàng Minh1, Phạm Minh Chiến1, Phạm Quốc Định1, Nguyễn Văn Thuận1,* Tóm tắt Sự đời cừu Dolly đánh dấu thành công nhân động vật kỹ thuật SCNT Đến nay, nhiều loài động vật nhân khỏe mạnh đời chuột, bò, khỉ Hơn nữa, tương lai kỹ thuật nhân vơ tính mang lại hiệu tiềm số lĩnh vực bảo tồn động vật quý hiếm, hỗ trợ trị liệu cho số bệnh người Do đó, nhiều nghiên cứu tập trung vào việc cải thiện thành công phương pháp tạo động vật nhân Nghiên cứu nhằm mục đích kiểm tra hiệu xử lý HDACi khoảng thời gian khác giai đoạn kích hoạt gen hợp tử (ZGA) với việc kết hợp loại HDACi Thí nghiệm hiệu việc xử lý HDACi với nồng độ khác phát triển phơi bị nhân Nhóm phơi xử lý với TSA nM + SCR 125 nM đạt tỷ lệ phát triển đến phôi nang cao (35,0 %) nhóm đối chứng nhóm TSA 10 nM + SCR 250 nM 21,7 % 21,1 % Tỷ lệ phơi nang bị nhân 20,0 % nhóm nM TSA + SCR 125 nM Thí nghiệm thứ hai tối ưu hóa thời gian xử lý giai đoạn ZGA cho bước thứ hai xử lý phơi bị nhân vơ tính Theo kết thu được, xử lý HDACi bước sau kích hoạt bước thứ hai kéo dài 10 tiếng từ lúc 52 h sau kích hoạt có 28,6 % phơi bị vơ tính phát triển đến giai đoạn phôi nang Kết cao đáng kể so với nhóm khác xem mốc thời gian tối ưu để xử lý HDACi cho phát triển phơi bị nhân Từ khóa: HDACi, nhân bị, kích hoạt gen hợp tử, SCNT, ZGA Trường Đại học Quốc tế, Đại học Quốc gia Thành phố Hồ Chí Minh Đóng góp nghiên cứu * Email: nvthuan@hcmiu.edu.vn # ... H4 acetylation of somatic chromatins in cloned embryos Journal of Biological Chemistry, 281: 6 048- 6057 Wilmut, I., A E Schnieke, J McWhir, A J Kind, and K H Campbell., 1997 Viable Offspring Derived