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.2022.0065 EFFECT OF CUMULUS CELL CO-CULTURED ON PREIMPLANTATION DEVELOPMENT OF CLONED BOVINE EMBRYOS Giang Trung Hieu1, Pham Truong Duy1, Mai Thi Quynh Nhu1, Tran Le Quy1, Nguyen Hoang Bao Ngan1, Lac Duong Hung1, Bui Hong Thuy1,*, Nguyen Van Thuan1,* Abstract This study examined the effects of the cumulus cell co-culture system on the development of cloned bovine embryos The results showed that the coculture of cloned bovine embryos with cumulus cells significantly improved the in vitro development rate to the blastocyst stage (25 %) compared to the control group (12.5 %) To evaluate the appropriate amount of cumulus cells used for supporting cloned bovine embryos development, an experiment that compared the growth of cumulus cells under embryo culture conditions found that a group of cumulus cells was more effective than a group of 10 cumulus cells based on the cell proliferation and attachment ability The current study demonstrated that the developmental competence and quality of cloned bovine embryos could be improved through in vitro co-culturing with cumulus cells during preimplantation development Keywords: Bovine, co-cultured, cumulus cells, embryos, in vitro, SCNT INTRODUCTION Since the first success of mammalian cloning was reported in 1997 (Wilmut et al., 1997), more than twenty species have been cloned by somatic cell nuclear transfer (SCNT) Within the advance of SCNT, cattle were one of the most attractive species for cloning research due to its potential application in agricultural and pharmaceutical production (Kim et al., 2009; Wu et al., 2012) Several in vitro culture systems resembling the bovine reproductive tract environment were established to improve the preimplantation development of cloned bovine embryos (Donnay et al., 1997; Rief et al., 2002) From previous studies, a positive effect on the quality and quantity of developing embryos has been observed with the addition of feeder layers or helper cells from various tissue types (fibroblasts, granulosa-cumulus cells) in comparison to standard culture conditions for in vitro (Farouk et al., 2008; Ju et al., 2012) Cumulus cells, in particular, are physiologically associated with the maturation of bovine oocytes and are immediately accessible following follicular aspiration of cumulus cells (CCs) This differentiation process commences simultaneously with follicular antrum formation, giving rise to two cell lineages: mural granulosa cells and CCs CCs are biologically distinct from other follicular cells, characterized by their distribution that appears in the form of cell layers encircling the oocytes The close proximity to the oocyte enables these cells to perform highly specialized communication with this germ cell (Gilchrist et al., 2008) * International University, Vietnam National University, Ho Chi Minh City Email: bhthuy@hcmiu.edu.vn, nvthuan@hcmiu.edu.vn PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 601 In such a communication process, trans-zonal cytoplasmic projections of cumulus cells play a pivotal role since they penetrate through the oocyte zona pellucida and connect with the oocytes by gap junctions that are formed at their tips (Ha et al., 2015) It was evident that the CCs oocyte’s bi-directional communication could bring significant benefits, in which the growth of somatic cells could be increased, and simultaneously, oocytes are appropriately nourished, enabling the enhancement in their developmental competence that ultimately leads to higher fetal viability To this extent, there raises the intriguing question of whether similar beneficial effects could be obtained when CCs or granulosa cells (GCs) are allowed to reside with cloned bovine embryos to form a coculture model that helps enhance the efficiency of cloning In this study, we examined the effect of co-culture cumulus cells on the preimplantation development of cloned bovine embryos, and proposed a new, simple approach for in vitro culturing cloned bovine embryos with a certain number of cumulus cells for obtaining cloned blastocyst at high quality and quantity MATERIALS AND METHOD 2.1 Collection of bovine cumulus – oocyte – granulosa cells complexes (COGCs) With their prior agreement, two slaughterhouses provided bovine ovaries, Ut Hao from Binh Duong province and Vissan from Ho Chi Minh City Ovaries were stored inside an insulated bottle containing Phosphate-buffered saline (PBS) The aspiration technique with a 10-cc syringe connected to an 18-gauge needle was used to collect cumulus-oocyte-granulosa cells complexes (COGCs) from early and large antral follicles (4-7 mm in diameter) Under a microscope, COGCs were obtained using a mouth pipette Collected COGCs were then culture in floating drop of 150 µL TCM 199 medium supplemented with 10 % fetal bovine serum (FBS), 0.1 mg/mL sodium pyruvate (Na-py), 0.05 mg/mL epidermal growth hormone (EGF), 0.1 IU/mL luteinizing hormone (LH), 0.01 IU/mL follicle- stimulating hormone (FSH), and 0.1 mg/mL estradiol Then incubated in 38.5 ºC and % CO2 in 20 hours for maturation 2.2 Collection and culture of bovine cumulus cells Cumulus cells in the third drops onwards yielded from the cumulus removal process were collected by mouth pipette, then transferred to a 1.5 mL tube containing HEPES for centrifugation The acquired dish was incubated at 38.5 oC, 5% CO2, allowing the cells to deposit on the bottom of the cell culture dish with DMEM + 10 % FBS The supplementation of the medium should be carried out gently to prevent cells from floating due to the force generated when rapidly releasing The dish was eventually incubated for the next growth phase and experiment 602 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM Figure Culture of bovine cumulus cells (A) Cumulus cell from oocyte cumulus cells complexes (B) CCs from the cumulus removal process (C) Start to adhere to the gelatin matrix (D) In elongated shape 2.3 Somatic cell nuclear transfer (SCNT) Enucleation was the first step performed in SCNT in order to take out the metaphase II chromosome of mature oocytes In details, a hole at the desired site on zona pellucida (ZP) was created by applying the XYClone laser (Hamilton Thorne), through which the enucleation pipette could enter to approach the oocyte membrane at the MII site and gently aspirate it out Enucleated oocytes were allowed to recover in the manipulating drop for 20 minutes before being gently rinsed in TCM free hormone medium, then transferred to a drop of the same medium and placed in the incubator During the oocyte’s recovery process, fibroblasts to be used for the subsequent injection were prepared Cell injection could be implemented one hour after the completion of enucleation When all oocytes have experienced nuclear transfer, they were left there for about 20 minutes before being rinsed with hormone-free TCM and allowed to recover in the same environment 2.4 Artificial activation and in vitro development (IVD) culture of reconstructed oocytes Artificial activation was conducted at 26 hours interval from the commencement of IVM culture All reconstructed oocytes entered the activation step with minutes in µM ionomycin (IO), instantly followed by additional hours in mM 6-dimethylaminopurine (6-DMAP) In addition, to lower epigenetic aberrance in SCNT embryos, a treatment of histone deacetylase inhibitors (HDACi) that last for 10 hours was applied after the activated oocytes had been washed in modified synthetic oviduct (mSOF) medium Eventually, all reconstructed oocytes were transferred to mSOF medium for in vitro development (IVD) culture in the incubator (38.5 oC, % CO2) 2.5 Co-culture of reconstructed oocytes with fibroblasts monolayer The first experiment examined the effect of IVD (5 % FBS) medium on the CCs growth The second experiment was divided into two groups, which were examined the preimplantation development of cloned bovine embryos in different culture conditions in terms of co-culture group, including without co-culture, and with cumulus cells monolayer coculture Co-culture was conducted on day from activation, using monolayer CCs that had been prepared in advance To create such a cell layer, the IVD culture dish was first prepared PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 603 in the form of droplets with 10 µL mSOF (5 % FBS) medium, covered by mineral oil to save them from evaporation Then, the culture dish was incubated at 38.5 oC and % CO2 After that, cumulus cells were harvested from the cell passage step and placed in the defined IVD drop All three groups experienced medium were changed by fresh medium at day RESULT AND DISCUSSION 3.1 The growth density of cumulus cells in the culture medium Collecting cumulus cells were cultured in drops 10 µL of mSOF medium (group without FBS and group with FBS) with different numbers of cells from the beginning (5 cells and 10 cells) of culture (Figure 2) Figure Cumulus cells after day in the culture medium with FBS (A) cumulus cells, (B) 10 cumulus cells Figure Cumulus cells after day in the culture medium without FBS (A) cumulus cells, (B) 10 cumulus cells Figure Cumulus cells after days in the culture medium with FBS (A) cumulus cells, (B) 10 cumulus cells The number of cells increases rapidly and fills the droplets (group 10 cumulus cells) Figure Cumulus cells after days in the culture medium without FBS (A) cumulus cells, (B) 10 cumulus cells Cumulus cells begin to attach to gelatin in the medium without FBS With a culture medium containing FBS, the number of the fibroblast was tightly attached to the gelatin surface, whereas the culture medium without FBS became floating in the culture medium Table Number of cumulus cells were cultured in drops 10 µL of mSOF medium with FBS Group Number of cells at Day Day Day Day Day Day Day 12(±1.5) 26(±2.1) 50(±2.5) 110 (±2.4) 220 (±2.3) 323 (±2.5) cells 10 27(±3.3) 60(±3.2) 135(±2.5) 314 (±2.1) 414 (±1.8) 432 (±1.8) 10 cells 604 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM This study used co-culture with cumulus cells to increase bovine cloning efficiency This experiment examined the growth density of cumulus cells in the culture medium and the effect of cumulus cell co-culture on the preimplantation development of cloned bovine embryos In the first results, the IVD medium with FBS enhanced the CCs develop (Figure 4A and 4B) Next, the number of cells in both experiments did not change after day in both FBS and non-FBS cultures (Figures and 3) In the culture medium containing FBS on day 7, the number of cells increased and divided in the group of cumulus cells (Figure 5A) and filled drops in the group of 10 cumulus cells (Figure 5B) In the culture medium without FBS, 10 cumulus cells showed signs of attachment surface to the cell culture dish The number of cumulus cells in the group of 10 CCs showed rapidly growing of cumulus cells in days (Table 1), but exhibited lower cell proliferation and attachment ability compared to the group of CCs (Figure 4A), in which the issue might directly affect the development of embryos because the number of cells was too much Thus, it led to insufficient nutrient medium for cells and embryos to develop together In contrast, the moderate number and growth of the group of cumulus cells ensured co-development with the embryo in the same drop in days 3.2 The effect of cumulus cell co-culture on preimplantation development of cloned bovine embryos Figure The obtained cloned bovine blastocysts (A) Control group (B) Cumulus cells co-culture group Scale bar: 100 µm Table Developmental rates of SCNT bovine embryos with cumulus cell co-cultured on preimplantation development of cloned bovine embryos Total Group embryos number 32 Control 32 Co-culture (3-5 cumulus cells) No (%) of the embryo at 2-cell 26(81.3) 28(87.5) 4-cell 20(62.5) 24(65.6) 8-cell 12(37.5) 19(59.4) Morula a 11(34.4) 12(37.5)a Blastocyst 4(12.5)a 8(25.0)b All experiments received HDACi treatment after chemical activation *Values with different superscripts within a column are significantly different (P < 0.05) This experiment had three replicates There were no significant differences between the co-culture treatment groups and the control group at 2-cell, 4-cell, 8-cell, and morula in term of developmental rates PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 605 although the treatment of cumulus cell co-cultured consistently showed higher percentages of embryos developed to each stage However, in the blastocyst formation rate, the cocultured group (25.0 %) was significantly higher than the control group (12.5 %) (Table 2) This result suggested that the cumulus cell co-cultured group had a positive impact on the development of cloned bovine embryos Moreover, an observation was made during this experiment that the group of cloned bovine embryos treated with cumulus cell cocultured reached to the later cell stages earlier and with higher percentages, morphology and quality compare with the control group CONCLUSION This study compared the growth of cumulus cells under embryo culture conditions and found that co-culture of cloned bovine embryos with cumulus cells was efficient for embryos development In conclusion, co-culture with cumulus cells significantly improved preimplantation development of cloned bovine embryos develop to blastocyst stage Acknowledgment: This study was funded by the Ministry of Science and Technology of Vietnam under grant number ĐTĐL.CN-49 Pham Truong Duy was funded by Vingroup Joint Stock Company and supported by the Domestic PhD Scholarship Programme of Vingroup Innovation Foundation (VINIF), Vingroup Big Data Institute (VINBIGDATA), code VINIF.2020.TS.27 REFERENCES Chian, R C., Lim, J H., & Tan, S L 2004 State of the art in in-vitro oocyte maturation Current Opinion in 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Co-culture (3-5 cumulus cells) No (%) of the embryo at 2-cell 26(81.3) 28(87.5) 4-cell 20(62.5) 24 (65. 6) 8-cell 12(37.5) 19(59.4) Morula a 11(34.4) 12(37.5)a Blastocyst 4(12.5)a 8(25.0)b All experiments... 2019 High cGMP and low PDE3A activity are associated with oocyte meiotic incompetence Cell Cycle, 18: 2629-2640 Gilchrist, R B., Lane, M & Thompson, J G., 2008 Oocyte-secreted factors: regulators