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 0072 IMPROVE PRONUCLEUS FORMATION IN ZYGOTE DERIVED FROM BOS GAURUS DEAD SPER[.]
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.2022.0072 IMPROVE PRONUCLEUS FORMATION IN ZYGOTE DERIVED FROM BOS GAURUS DEAD SPERM AND BOS TAURUS SMALL OOCYTES BY PRE-MATURATION CULTURE Cao Hoang Nam1, Dao Quang Tri1, Pham Truong Duy1, Lam Do Truc Phuong1, Nguyen Van Thuan1, Bui Hong Thuy1,* Abstract Growing oocytes derived from small antral follicles (2-3 mm) have poor maturation rates and developmental competence compared to fully grown oocytes collected from large antral follicles (4-6 mm) Hence, a pre-in vitro maturation (Pre-IVM) subculture with in vitro maturation (IVM) was applied to support small oocytes growth The objective of this study is to examine the effects of Pre-IVM culture on the developmental competence of growing oocytes Moreover, this study also investigated the ability to form male pronucleus from dead sperm of growing oocytes after Pre-IVM culture Therefore, the maturation rate of growing oocytes with and without Pre-IVM culture was recorded in the first experiment After that, the total cell number of parthenogenetic embryos on day seven was checked to evaluate the potential of the Pre-IVM culture system Finally, the intracytoplasmic sperm injection (ICSI) technique injected Bos Gaurus dead sperm into Bos Taurus growing oocytes was carried out to investigate male pronuclear formation in growing oocytes with Pre-IVM and IVM culture As a result, the maturation rate of growing oocytes improved from 34 % to 76 % after treatment with Pre-IVM In addition, the Pre-IVM culture improved the proportion of high-quality parthenogenetic embryos with more than 80 cells up to 50 %, while without Pre-IVM cannot obtain any high quality blastocyst like this Moreover, we obtained 17 % of zygotes in the pre-IVM treatment group successfully formed male pronucleus, while bull sperm heads remained arrested in all growing oocytes derived from small antral follicles without pre-IVM treatment In conclusion, Pre-IVM improved maturation rate and developmental competence, and equipped the ability to form male pronucleus from dead sperm to growing oocytes after IVM Keywords: Hybrid embryos, intracytoplasmic sperm injection, parthenogenetic embryos, pre-in vitro maturation, pronucleus formation INTRODUCTION In 2015, a dead male gaur’s testis had been cryopreserved in the Cellular Reprogramming Laboratory at International University, Vietnam National University of HCM city, Vietnam for research purposes Because gaur’s sperm were dead, the sperm could not participate in the in vitro fertilization (IVF) process Thus, intracytoplasmic sperm injection (ICSI), which involves the fertilization of oocytes in the metaphase II stage by direct injection of the spermatozoon (Goto et al., 1997), can be a powerful tool and suitable choice for scientists to recover the population of wild gaur Due to the low * International University, Vietnam National University, Ho Chi Minh City Email: bhthuy@hcmiu.edu.vn 656 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM population of wild gaur, collecting oocytes from female gaur for ICSI experiments can become an obstacle In the native breeding, a domestic bovine, Bos Taurus (2n = 60), could be hardly fertilized with a gaur (2n = 56) Therefore, the inter-species ICSI method by injecting gaur sperm into Bos Taurus oocytes can be a solution for having a new breed In vitro maturation (IVM) is a procedure that has been broadly applied in recovering immature oocytes to fully meiotic competence However, the number of fully grown oocytes collected for IVM culture from each ovary was limited, while growing oocytes are abundant and can be considered alternative source material for the ICSI technique On the other hand, growing oocytes derived from small antral follicles (2-3 mm) cultured immediately in IVM medium right after being collected showed poor maturation rates and low developmental competence due to its incomplete in both cytoplasm and nucleus (Lonergan et al., 1994) By inhibiting germinal vesicle breakdown (GVBD) and keeping growing oocytes at the germinal stage (GV) before IVM, the Pre-IVM technique allows oocytes to acquire complete developmental competence during the meiotic arrest Lascorbic acid is an essential micronutrient known for the ability of modulating many biochemical processes as an antioxidant Some study demonstrated that L-ascorbic acid enhance the developmental competence of porcine oocytes after fertilization (Tatemoto et al., 2001) Our group also demonstrated that L-ascorbic acid could improve the developmental competence of embryo during preimplantation through histone modifications in porcine oocytes from small antral follicles (Thuy Van et al., 2020) In addition, in bovine, our group also indicated that the Pre-IVM medium supplemented with 50 µg/mL L-ascorbic acid increased meiotic resumption, developmental competence, and embryo quality derived from growing oocytes collected from 2-3 mm follicles (unpublished data) Thus, growing oocytes treated with Pre-IVM could be a potential material for the inter-species ICSI to conserve endangered gaur Besides that, the success rate of ICSI in the bovine is low compared to the rate of embryo development obtained by IVF Specifically, in this species, most oocytes fail to activate following ICSI (Malcuit et al., 2006) and display delayed and incomplete sperm head decondensation (Suttner et al., 2000) Various stimuli for oocyte activation, such as cycloheximide (Rho et al., 1998), and calcium ionophores (Bevacqua et al., 1998), have been used to activate reconstructed bovine oocytes after the ICSI procedure artificially Artificial activation can also lead to unwanted haploid embryo formation if the cytoplasm of the oocytes fails to decondense the sperm head Therefore, determining the first cell cycle of inter-species zygotes is a foundation for investigating the effect of pre-IVM on the cytoplasmic maturation of small oocytes In this study, we first determined the effect of Pre-IVM culture on the development of growing oocytes derived from 2-3 mm follicles Then, we produced inter-species zygotes by injecting a single dead Bos Gaurus sperm into these Bos Taurus oocytes and then observed the first cell cycle of the zygote to evaluate the pre-IVM culture system on small oocytes PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 657 MATERIALS AND METHODS 2.1 Collection of OCGCs from small antral follicles (2-3 mm) and Pre-IVM culture Bovine ovaries were delivered from slaughterhouses to Cellular Reprogramming Laboratory in phosphate-buffered saline (PBS) container After washing with PBS, performing dissection method, the surface of ovaries was cut to obtain 3-4 mm thick slices Slices were washed through PBS-PVA 0.1 % and continued to be dissected to collect 2-3 mm follicles Good quality follicles then were stored in HEPES solution After that, follicles were torn with forceps and needles in HEPES solution to collect Oocytescumulus-granulosa-complexes (OCGCs) High-quality OCGCs were transferred to the HEPES dish The OCGCs were cultured in floating drops of Pre-IVM medium for 13 h at 38.5 oC in humidified air with % CO2 The Pre-IVM medium was αMEM supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL sodium pyruvate, mM hypoxanthine, 0.01 IU/mL follicle-stimulating hormone (FSH), 10 ng/mL androgen, µg/mL estradiol17β and 50 µg/mL L-ascorbic acid After hours of Pre-IVM culture, 1.5 µL human chorionic gonadotropin (hCG) hormone (0.1 IU/mL) was supplied to each floating drop 2.2 Collection of OCGCs from antral follicles (4-6 mm) and IVM culture OCGCs were isolated from 4-8 mm large follicles using the aspiration method OCGCs isolated were washed several times with HEPES to remove the uncollected tissues, then cultured in floating drops of IVM medium as the control group After PreIVM culture, OCGCs were transferred to floating drops of IVM medium which contained TCM-199 medium supplemented with 10 % FBS, 10 µg/mL Sodium pyruvate, 50 ng/mL Epidermal Growth Factor (EGF), g/mL FSH, 0.0065 IU/mL Luteinizing hormone (LH) and µg/mL Estradiol for 19 h at 38.5 oC in humidified air with 5% CO2 2.3 Intracytoplasmic sperm injection (ICSI) After IVM culture, hyaluronidase was added into IVM floating drops Cumulus cells were removed by mouth pipette in HEPES, and only matured oocytes with the first polar body were selected for ICSI Matured oocytes were recovered in TCM-free hormone that contains TCM-199 supplemented with 10 % FBS and 10 µg/mL sodium pyruvate for half an hour A small number of oocytes (5-10) were manipulated at a time, whereas the remaining oocytes were maintained in the recovery medium ICSI was performed at 400× magnification in a drop of HEPES under mineral oil on a Nikon 200 inverted microscope stage After that, injected oocytes continue to be recovered in TCM-free hormone for 30 minutes and then transferred into the drop of modified synthetic oviduct fluid medium (mSOF) for hours at 38.5 oC in humidified air with % CO2 2.4 Artificial activation Oocytes were activated in mSOF containing µM calcium ionophore ionomycin for minutes Oocytes were placed in a drop of mSOF for three hours to allow second polar body extrusion After that, oocytes were treated with 6-dimethylaminopurine for hours Oocytes were then washed with mSOF culturing in mSOF for in vitro development (IVD) under optimal conditions of 38.5 oC and % CO2 658 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM 2.5 Immunostaining Zygotes at h, and 14 h after activation were fixed in % paraformaldehyde for 4045 minutes and then soaked in Triton x-100 at oC overnight These zygotes were immersed in the first antibody (rabbit polyclonal anti-trimethyl-histone H3 at lysine (H3K9me3) and mouse monoclonal anti-lamin B for hours Then, using the second antibody (Alexa flour 488-labeled chicken anti-rabbit IgG and Alexa flour 568 labeled goat anti-mouse immunoglobulin IgG antibodies) for hour Finally, they were counterstained by 4’,6-diamidino-2-Phenylindole dihydrochloride (DAPI) for 30 minutes Samples were mounted on microscopic glass using glycerol covered with a glass coverslip to observe under fluorescence microscope which was a Nikon Inverted Microscope Eclipse Ti-U 2.6 Statistical analysis Statistical analysis was performed by SPSS 20 The data were labeled by singlefactor analysis of variance (ANOVA) followed by the Turkey test Each group of experiments was repeated to times to ensure the statistical power of the test P values less than 0.05 were considered statistically significant For quantitative analyses, the fluorescent images were subjected to densitometric analysis using NIS Elements BR software RESULT AND DISCUSSION 3.1 The effect of Pre-IVM culture on meiotic resumption of growing oocytes Figure Meiotic competence of bovine oocytes after 19 h culturing in IVM a, b Different letters indicate a significant difference (P < 0.05) After culturing in the IVM medium, the maturation rate of the growing oocyte group was recorded and compared to the fully grown oocyte group As shown in Figure 1, the maturation rate of growing oocytes without Pre-IVM was only 34.26 % ± 5.24 % (n = 33), while the proportion of the group treated with Pre-IVM was similar to the fully grown oocytes group (78.34 % ± 3.84 % (n = 28) and 76.49 % ± 5.76 % (n = 37), respectively) This figure demonstrated that Pre-IVM culture improved the meiotic competence of growing oocytes collected from the small antral follicles (2-3 mm) PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 659 3.2 The effect of Pre-IVM culture on developmental competence of parthenogenetic embryos derived from growing oocytes Figure Quality of bovine parthenogenetic blastocyst based on cell number counting (A) Parthenogenetic embryo quality bar chart (B) DAPI staining picture a ,b Different letters indicate a significant difference among good blastocyst groups (P < 0.05) x ,y Different letters indicate a significant difference among average blastocyst groups (P < 0.05).* ,** ,*** Different letters indicate a significant difference among poor blastocyst groups (P < 0.05) After culturing in IVD for 7.5 days, bovine parthenogenetic blastocysts were fixed and stained with DAPI By counting the total number of cells, qualification analysis of blastocysts could be determined The quality of bovine blastocysts was arranged into three categories: poor (30-50 cells), average (50-80 cells), and good (> 80 cells), which was shown in Figure The majority of the blastocyst group without Pre-IVM culture was of poor quality (63 %), and no blastocyst reached over 80 cells On the other hand, compared to the group that underwent Pre-IVM culture, the blastocyst quality was higher than the group without Pre-IVM culture, improving the percentage of good blastocyst from % to 55 % and lowering the proportion of poor blastocyst from 62 % to 15 % To sum up, this data indicated that oocytes that underwent Pre-IVM culture improved the quality of bovine parthenogenetic blastocysts derived from the small antral follicles 3.3 The effect of Pre-IVM culture of growing oocytes on male pronucleus formation in inter-species zygotes As shown in Figure 3, immune-fluorescence staining with H3K9me3 antibody was used to confirm the identity of the female component (maternal nucleus and two polar bodies) as H3K9 was demethylated on the male pronuclear The red circle indicated male pronucleus location while the red arrow indicated arrested sperm head location After h post- activation, sperm decondensed in the fully grown oocytes group In the next h, the paternal nucleus showed bigger than the maternal pronuclear However, in the growing oocytes group without Pre-IVM culture, sperm was arrested and failed to form pronuclear in all oocytes Thus, the maternal nucleus underwent mitotic division and expressed the H3K9 methylation signal This result meant all oocytes in this group became parthenogenetic embryos In Figure 4, the zygote derived from growing oocytes treated with Pre-IVM forms the male pronucleus successfully Lamin B signals indicate the 660 BÁO CÁO KHOA HỌC VỀ NGHIÊN CỨU VÀ GIẢNG DẠY SINH HỌC Ở VIỆT NAM location of two pronuclei, and the male pronucleus expressed histone demethylation similar to those derived from fully grown oocytes Figure Immunostaining for male pronucleus detection in inter-species zygotes of fully grown oocytes group and growing oocytes without Pre-IVM Figure Immunostaining for male pronucleus detection in inter-species zygotes of growing oocytes with Pre-IVM Figure The proportion of male pronucleus formation after injecting Bos Gaurus dead sperm into Bos Taurus oocytes a ,b ,c Different letters indicate a significant difference (P < 0.05) As shown in Figure 5, when examined the inter-species embryos at h and 14 h post- activation, over 63 % of sperm decondensed and formed pronuclear in all sperm injected fully grown oocytes group However, in the growing oocytes group without PreIVM culturing, sperm were arrested in all oocytes On the other hand, when culturing growing oocytes with Pre-IVM, a small proportion of these oocytes (17 %) can decondense sperm heads This result demonstrated that growing oocytes derived from small antral follicles need to undergo Pre-IVM culture to achieve the ability to form male pronucleus from injected dead sperm PHẦN NGHIÊN CỨU ỨNG DỤNG SINH HỌC PHỤC VỤ ĐỜI SỐNG VÀ PHÁT TRIỂN XÃ HỘI 661 CONCLUSION In conclusion, this study demonstrated that the Pre-IVM sub-culture system could increase the development of growing oocytes as high as the fully grown ones It also indicated that growing oocytes need to undergo Pre-IVM culture to have the ability to decondense dead sperm head into male pronuclear However, the ability to convert dead sperm head into male pronuclear was not high compared to fully grown oocytes Therefore, this sub-culture system still needs to be researched more to apply to endangered gaur reservation programs in the future Acknowledgements: This research is funded by Vietnam Ministry of Science and Technology under Grant No ĐTĐL.CN-49/16 REFERENCES Bevacqua, R., Canel, N., Fernandez-Martin, R., and Salamore, D F., 1998 Activation with Ionomycin followed by Dehydroleucodine and Cytochalasin B for the Production of Parthenogenetic and Cloned Bovine Embryos Cellular Reprogramming, 12(4): 491-499 Goto, K., 1997 Current status and future of micromanipulation-assisted fertilization in animals and human Journal of Reproduction and Development, 43(2): 107-119 Lonergan, P., Monaghan, P., Rizos, D., Boland, M P., and Gordon, I., 1994 Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro Molecular Reproduction & Development, 37(1): 48-53 Malcuit, C., Kurokawa, M., and Fissore, R A., 2006 Calcium oscillations and mammalian egg activation Journal of Cellular Physiology, 206(3): 565-573 Rho, G J., Kawarsky, S., Johnson, W H., Kochhar, K., and Betteridge, K J., 1998 Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes Biology of Reproduction, 59(4): 918-924 Suttner, R., Zakhartchenko, V., Stojkovic, P., Muler, S., Alberio, R., Medijugorac, I., Brem, G., Wolf, E., and Stojkovic, M., 2000 Intracytoplasmic sperm injection in bovine: Effects of oocyte activation, sperm pretreatment and injection technique Theriogenlogy, 54(6): 935-948 Tatemoto, H., Ootaki, K., Shigeta, K., and Muto, N., 2001 Enhancement of Developmental Competence after In Vitro Fertilization of Porcine Oocytes by Treatment with Ascorbic Acid 2-O-α-Glucoside During In Vitro Maturation Biology of Reproduction, 65(6): 1800-1806 ThuyVan, N T., My L B A., Thuan, N V., and Bui, H T., 2020 Improve the developmental competence of porcine oocytes from small antral follicles by prematuration culture method Theriogenology, 149: 139-148