Journal of Animal Reproduction and Biotechnology 2022; 37(1): 48-54
Published online March 31, 2022
https://doi.org/10.12750/JARB.37.1.48
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Bitnara Kim1 , Seongjun So1
, Jiwan Choi2
, Eunju Kang1,2,*
and Yeonmi Lee1,2,*
1Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Korea
2Center for Embryo and Stem Cell Research, CHA Advanced Research Institute, CHA University, Seongnam 13488, Korea
Correspondence to: Yeonmi Lee
E-mail: yeonmilee82@chamc.co.kr
Eunju Kang
E-mail: ekang@cha.ac.kr
Parthenogenesis is maternally uniparental reproduction through the embryonic development of oocytes without fertilization. Artificial activation of mature oocytes could generate homozygous haploid embryos with the extrusion of the second polar body. However, the haploid embryos showed low embryo development in preimplantation embryos. In this study, we investigated whether the electronic fusion of the haploid embryos could enhance embryo development and ESC establishment in mice. Haploid embryos showed the developmental delay from 4-cell to the blastocyst stage. The haploid blastomeres of the 2-cell stage were fused electronically, resulting in that the fused embryos showed a significantly higher rate of blastocysts compared to non-fused haploid embryos (55% vs. 37%). Further, the embryonic stem cells (ESCs) derived from the fused embryos were confirmed to be diploid. The rate of ESC establishment in fused embryos was significantly higher compared to non-fused ones. Based on the results, we concluded that the electronic fusion of haploid embryos could be efficient to generate homozygous ESCs.
Keywords: embryonic stem cells, homozygous, parthenogenesis, 2-cell fusion
Parthenogenesis refers to maternally uniparental reproduction, which is a reproduction strategy through the embryonic development of oocytes without fertilization (Daughtry and Mitalipov, 2014; Jung et al., 2020). Parthenogenesis is not a natural form of reproduction in mammals. The birth of offspring from parthenogenetic embryos is not possible because of the lack of expression of imprinted paternal genes, which are required for the generation of the functional placenta (McGrath and Solter, 1984). This characteristic allows the application of parthenogenetic embryonic stem cells (pESCs), avoiding the ethical issues with biparental ESCs. Further, pESCs show self-renewal and pluripotent differentiation ability, which are similar characteristics to biparental ESCs (Didi? et al., 2013).
In the experiments for the artificial activation of mature oocytes, the activated oocytes, inhibited the extrusion of the second polar body (the 2nd PB), could generate pseudodiploid embryos (Lee and Kang, 2019). These parthenogenetic oocytes can harbor unseparated sister chromatids, enter mitosis, and produce partially heterozygous embryos due to chromosome crossover. These embryos showed a similar developmental rate into blastocysts compared to fertilized embryos (Mitalipov et al., 2001). When the activated oocytes extrude the 2nd PB similar to natural activation, these parthenogenetic oocytes harbor the separated sister-chromatid and finally produce haploid embryos with homozygosity (Lee and Kang, 2019). These embryos can be established to haploid ESCs, which could be a valuable source for genetic screening and cell therapy (Wutz, 2014; Ding et al., 2015). For the genetic screening, diploid cells could have heterozygous mutations, which could show few or no phenotypic changes in recessive mutations (Wutz, 2014). However, the mutations that existed in a haploid genome, a hemizygous state, can result in the complete exposure of phenotypes in the cells. Haploid cells show homozygosity for the human leukocyte antigen (HLA) type, which could be a useful source for cell therapy by preventing immune rejection in allografts (Ding et al., 2015).
The pESCs are considered a valuable source to study the influence of paternal imprinting. There is a limitation to the full-term development of parthenogenetic embryos due to their genomic imprinting status (McGrath and Solter, 1984). A recent study demonstrated that the modification of imprinted genes in oocytes could produce viable offspring from the parthenogenetic embryos (Wei et al., 2022).
Previous studies reported that the haploid embryos showed impaired development in preimplantation embryos compared to diploid embryos (Witkowska, 1973; Kaufman and Sachs, 1976; Latham et al., 2002). To enhance the development of haploid embryos, we hypothesized that the electronic fusion of blastomeres in a haploid embryo could induce diploid, which can result in an increased rate of embryo development and ESC establishment.
In this study, we analyzed the embryo development of the parthenogenetic haploid embryos in mice and investigated that electronic fusion of the haploid embryo could increase the rate of embryo development and homozygous ESC establishment.
B6D2F1 (C57BL/6N female × DBA2 male) female (8 to 9-week-old, Charles River) were used for the experiment. All animal maintenance and experimental procedures were performed following the guidelines of CHA University. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No.210035) of CHA University.
The experiment related to MII oocyte collection was performed according to the previously described methods with minor modification (Kang et al., 2014; Kim and Lee, 2019; So et al., 2020). Female mice were super-ovulated with 5 international units (IU) pregnant mare’s serum gonadotropin (PMSG), followed by 5 IU human chorionic gonadotropin (hCG) 48 h later. MII oocytes were collected 16 h after hCG injection. The denuded oocytes by 0.1% hyaluronidase solution (Sigma) were kept in KSOM (Merck) at 37℃ in a humidified incubator with 5% CO2.
Oocytes were incubated in an activation medium supplemented with 10 mM SrCl2 (Sigma) in the presence or absence of 5 ug/mL cytochalasin B (CB, Sigma) for six hours at 37℃ in a humidified incubator with 5% CO2. After activation, the oocytes were examined for the formation of pronucleus (PN). Activated oocytes were cultured for four to five days to the blastocysts in KSOM (Merck) at 37℃ in a humidified incubator with 5% CO2 (Lee and Kang, 2021).
2-cell embryos are placed between the electrodes of a 100 ?m gap electrode chamber (BLS Ltd.) containing 0.28 M Mannitol (Sigma), 0.1 mM magnesium sulfate hydrate (Sigma), 50 ?M calcium chloride (Sigma), and 0.3% bovine serum albumin (Sigma) and fused by Electro Cell Manipulator ECM 2001 (BTX). An initial electrical field of 2V is applied to the embryos followed by one peak pulse of 50V for 35 ?s. After fusion, the embryos were transferred to KSOM and observed for fusion.
Denuded blastocysts were placed onto mitomycin C (Sigma)-treated mouse embryonic fibroblast (MEF) feeder in mESC medium: KODMEM (Gibco) containing 20% KOSR (Gibco), 1 mM L-glutamine (Gibco), 100 units/mL penicillin (Hyclone), 100 ?g/mL streptomycin (Hyclone), 100 ?M β-mercaptoethanol (Sigma), 100 ?M nonessential, and 1,000 units/m LIF (Stemgent) under 5% CO2 at 37℃ in a humidified incubator. The cell outgrowth was replaced on the new MEF feeder. mESCs were passaged every 3 to 4 days for further experiments.
Cells were fixed with 70% ethanol at 4℃ for 15 min and stained with 50 ?g/mL propidium iodide (Sigma) solution containing 100 ?g/mL RNase A at 4℃ for 40 min. The cell cycle was measured by a FACS Calibur (BD Biosciences) and the data were analyzed by FlowJo v10.5.3 (FlowJo LLC).
KaryoMAX Colcemide (Gibco) with 150 ng/mL concentration was applied to the cultured ESCs for 1.5 h at 37℃. The treated cells were detached by trypsin/EDTA. The cells treated with 0.075 M KCL hypotonic solution for 30 min were fixed with methanol: acetic acid (3:1 v/v) and dropped onto a slide. The slides were mounted in Prolong Diamond Antifade Mountant with DAPI (Invitrogen).
Data were analyzed with Fisher’s exact test for two groups and ANOVA with Tukey analysis for multiple comparisons. Statistical analyses were performed using GraphPad Prism (version 6). Data are expressed as mean ± standard error of the mean (S.E.M), and
To produce diploid parthenogenic embryos by activation, MII oocytes were activated with CB, resulting in that the zygotes could show two pronuclei (2 PNs) and no extrusion of the 2nd PB after activation (Bałakier and Tarkowski, 1976) (SrCl2+CB group in Fig. 1A). While, for the generation of haploid embryos, MII oocytes were activated without CB, and the zygote could show one pronucleus (1 PN) and the extrusion of the 2nd PB (SrCl2-CB group in Fig. 1A). Some haploid blastomeres on the 2-cell stage were performed electronic fusion to induce diploidy (SrCl2-CB with 2-cell fusion group in Fig. 1A).
After activation, the PN formation rate in the SrCl2+CB and SrCl2-CB group was comparable (100% vs. 97%) (Fig. 1B). Both groups showed the embryo development to blastocyst (Fig. 1C). The embryo development from 2-cell to morula stage was similar between the SrCl2+CB and SrCl2-CB group (100% vs. 96% for 2-cell; 100% vs. 89% for 4-cell; 100% vs. 91% for morula). However, the blastocyst rate was significantly lower in an SrCl2-CB group than in the SrCl2+CB group (37% vs. 100%) (Fig. 1B).
When the 2-cell embryos in the SrCl2-CB group were fused electronically, the embryos were changed their morphology to one cell, cleaved again on the next day, and developed into blastocysts (Fig. 1C). The embryo development from 2-cell to morula stage was 86% for 2-cell, 94% for 4-cell, and 91% for morula in SrCl2-CB with 2-cell fusion group, which was comparable to SrCl2+CB and SrCl2-CB group (Fig. 1B). However, the blastocyst rate was significantly higher in the SrCl2-CB with 2-cell fusion group than in the SrCl2-CB group (55% vs. 37%), which was significantly lower than in the SrCl2+CB group (100%) (Fig. 1B).
During the embryo development, the blastocysts in the SrCl2+CB group were formed on day 4 and hatched on day 5 (Fig. 1C). While, in the SrCl2-CB group, the blastocysts were started to appear on day 5 and the quality of blastocyst was worse compared to the SrCl2+CB group (Fig. 1C). Based on this result, we examined what embryo stage was delayed during the embryo culture in the SrCl2-CB group. We compared the distribution of 2-cell at 32 hours, 4-cells at 56 hours, morula at 80 hours, and blastocyst at 120 and 128 hours in the cultured embryos of SrCl2+CB group, SrCl2-CB group, and SrCl2-CB with 2-cell fusion group (Fig. 1D). There was no significant difference in 2-cell distribution at 32 hours between the SrCl2+CB group and SrCl2-CB group. However, the distribution of 4-cells at 56 hours, morula at 80 hours, and blastocyst at 120 and 128 hours was significantly lower in the SrCl2-CB group compared to the SrCl2+CB group. In SrCl2-CB with 2-cell fusion group, the distribution of morula at 80 hours, and blastocyst at 120 and 128 hours was significantly higher than SrCl2-CB group but lower than SrCl2+CB group. Based on results, the SrCl2-CB group showed developmental delay from 4-cell to blastocyst stage while SrCl2-CB with 2-cell fusion group displayed the improved development delay.
ESCs were established from the blastocysts in each group and the cell cycle analysis was performed (Fig. 2A and 2B). The mouse fibroblasts were used as a diploid control. ESCs in SrCl2+CB and SrCl2-CB with 2-cell fusion group were confirmed the diploid (Fig. 2B). While ESCs in the SrCl2-CB group displayed haploid. We confirmed the haploid in the SrCl2-CB group and diploid in SrCl2-CB with the 2-cell fusion group by karyotyping (Fig. 2C). The rate of ESC establishment from the blastocysts was significantly lower in the SrCl2-CB group compared to SrCl2+CB and SrCl2-CB with 2-cell fusion group (5.3% vs. 78.3% and 51.5%) (Fig. 2D). The rate in SrCl2-CB with 2-cell fusion group was lower than SrCl2+CB group but was not a significant difference. When the rate of ESC establishment was calculated based on the number of MII oocytes, the SrCl2-CB group showed a significantly lower rate than SrCl2+CB and SrCl2-CB with 2-cell fusion group (1.4% vs. 78.3% and 20%) (Fig. 2D). The rate in SrCl2-CB with the 2-cell fusion group was significantly lower than in the SrCl2+CB group. These results suggested that electronic fusion could increase the rate of homozygous ESC establishment.
The present study demonstrated whether the electronic fusion of haploid embryos could induce the increase of homozygous ESCs establishment. Electronic fusion of haploid embryos induced significantly higher blastocyst generation and ESC establishment compared to non-fused ones.
In mice, parthenogenetic embryos usually maintained haploidy at the blastocyst stage (Tarkowski et al., 1970; Kaufman et al., 1983), however, post-implantation haploid embryos tended to become diploid
Previous studies demonstrated that haploid embryos showed a lower developmental rate in preimplantation embryos than the diploid embryos (Witkowska, 1973; Kaufman and Sachs, 1976; Latham et al., 2002), which was also observed in our study. Further, we demonstrated that the developmental delay of haploid embryos occurred in 4-cell to the blastocyst stage. Electronic fusion was performed in the haploid 2-cell, resulting in the blastocyst rate being significantly increased compared to non-fused embryos. Further, the rate of ESCs establishment was higher in fused embryos than non-fused one, suggesting more homozygous ESCs could be generated due to the higher efficiency of blastocyst and ESC generation.
Electronic fusion of 2-cell blastomeres is often used for tetraploid complementation (Gertsenstein, 2015). When we applied the same method in haploid 2-cells, the embryo development of 2-cell to morula was comparable to the non-fused haploid embryo, further the blastocyst rate was significantly higher, resulting in the electronic fusion did not damage the embryo development and quality in haploid embryos.
There is another method to produce parthenogenic haploid embryos, which involves removing the male PN from the fertilized zygote (Bai et al., 2016). However, this method requires complex processes, such as fertilization and removing PN by manipulation, and is hard to apply in humans due to the ethical issue of destroying the fertilized embryos. Therefore, the activation of mature oocytes could be a simple and efficient method to produce a parthenogenetic haploid embryo. Further, the electronic fusion of the haploid embryo could induce a higher rate of blastocyst development, which could lead to higher efficiency for the generation of ESCs with homozygosity.
Haploidization of the diploid somatic genome was tried using mouse oocytes (Tateno et al., 2003; Lee et al., 2022). The method described in the current article could be applied in somatic haploid embryos to improve the establishment of somatic homozygous ESCs.
Haploid embryos showed a lower blastocyst rate and the developmental delay from 4-cell to blastocyst stage compared to diploid embryos. When the electronic fusion was performed in haploid embryos, the developmental delay was improved. The rates of blastocyst development and ESC establishment were significantly increased compared to the non-fused group. Based on the result, we concluded that the electronic fusion of haploid embryos could be an efficient method to generate homozygous ESCs.
None.
Conceptualization, Y.L., E.K.; data curation, B.K., Y.L.; formal analysis, B.K., S.S., J.C., Y.L.; funding acquisition, Y.L., E.K.; investigation, B.K., S.S., J.C., Y.L.; methodology, B.K., S.S., J.C., Y.L.; project administration, Y.L., E.K.; resources, B.K., Y.L.; software, B.K., J.C., Y.L.; supervision, Y.L., E.K.; validation, B.K., S.S., J.C., Y.L.; visualization, B.K., Y.L.; writing - original draft, B.K.; writing - review & editing, Y.L., E.K.
This work has been supported by the National Research Foundation of Korea (grant nos. NRF-2018R1A2B3001244 and NRF-2021R1I1A1A01049705).
The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No.210035) of CHA University.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2022; 37(1): 48-54
Published online March 31, 2022 https://doi.org/10.12750/JARB.37.1.48
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Bitnara Kim1 , Seongjun So1
, Jiwan Choi2
, Eunju Kang1,2,*
and Yeonmi Lee1,2,*
1Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Korea
2Center for Embryo and Stem Cell Research, CHA Advanced Research Institute, CHA University, Seongnam 13488, Korea
Correspondence to:Yeonmi Lee
E-mail: yeonmilee82@chamc.co.kr
Eunju Kang
E-mail: ekang@cha.ac.kr
Parthenogenesis is maternally uniparental reproduction through the embryonic development of oocytes without fertilization. Artificial activation of mature oocytes could generate homozygous haploid embryos with the extrusion of the second polar body. However, the haploid embryos showed low embryo development in preimplantation embryos. In this study, we investigated whether the electronic fusion of the haploid embryos could enhance embryo development and ESC establishment in mice. Haploid embryos showed the developmental delay from 4-cell to the blastocyst stage. The haploid blastomeres of the 2-cell stage were fused electronically, resulting in that the fused embryos showed a significantly higher rate of blastocysts compared to non-fused haploid embryos (55% vs. 37%). Further, the embryonic stem cells (ESCs) derived from the fused embryos were confirmed to be diploid. The rate of ESC establishment in fused embryos was significantly higher compared to non-fused ones. Based on the results, we concluded that the electronic fusion of haploid embryos could be efficient to generate homozygous ESCs.
Keywords: embryonic stem cells, homozygous, parthenogenesis, 2-cell fusion
Parthenogenesis refers to maternally uniparental reproduction, which is a reproduction strategy through the embryonic development of oocytes without fertilization (Daughtry and Mitalipov, 2014; Jung et al., 2020). Parthenogenesis is not a natural form of reproduction in mammals. The birth of offspring from parthenogenetic embryos is not possible because of the lack of expression of imprinted paternal genes, which are required for the generation of the functional placenta (McGrath and Solter, 1984). This characteristic allows the application of parthenogenetic embryonic stem cells (pESCs), avoiding the ethical issues with biparental ESCs. Further, pESCs show self-renewal and pluripotent differentiation ability, which are similar characteristics to biparental ESCs (Didi? et al., 2013).
In the experiments for the artificial activation of mature oocytes, the activated oocytes, inhibited the extrusion of the second polar body (the 2nd PB), could generate pseudodiploid embryos (Lee and Kang, 2019). These parthenogenetic oocytes can harbor unseparated sister chromatids, enter mitosis, and produce partially heterozygous embryos due to chromosome crossover. These embryos showed a similar developmental rate into blastocysts compared to fertilized embryos (Mitalipov et al., 2001). When the activated oocytes extrude the 2nd PB similar to natural activation, these parthenogenetic oocytes harbor the separated sister-chromatid and finally produce haploid embryos with homozygosity (Lee and Kang, 2019). These embryos can be established to haploid ESCs, which could be a valuable source for genetic screening and cell therapy (Wutz, 2014; Ding et al., 2015). For the genetic screening, diploid cells could have heterozygous mutations, which could show few or no phenotypic changes in recessive mutations (Wutz, 2014). However, the mutations that existed in a haploid genome, a hemizygous state, can result in the complete exposure of phenotypes in the cells. Haploid cells show homozygosity for the human leukocyte antigen (HLA) type, which could be a useful source for cell therapy by preventing immune rejection in allografts (Ding et al., 2015).
The pESCs are considered a valuable source to study the influence of paternal imprinting. There is a limitation to the full-term development of parthenogenetic embryos due to their genomic imprinting status (McGrath and Solter, 1984). A recent study demonstrated that the modification of imprinted genes in oocytes could produce viable offspring from the parthenogenetic embryos (Wei et al., 2022).
Previous studies reported that the haploid embryos showed impaired development in preimplantation embryos compared to diploid embryos (Witkowska, 1973; Kaufman and Sachs, 1976; Latham et al., 2002). To enhance the development of haploid embryos, we hypothesized that the electronic fusion of blastomeres in a haploid embryo could induce diploid, which can result in an increased rate of embryo development and ESC establishment.
In this study, we analyzed the embryo development of the parthenogenetic haploid embryos in mice and investigated that electronic fusion of the haploid embryo could increase the rate of embryo development and homozygous ESC establishment.
B6D2F1 (C57BL/6N female × DBA2 male) female (8 to 9-week-old, Charles River) were used for the experiment. All animal maintenance and experimental procedures were performed following the guidelines of CHA University. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No.210035) of CHA University.
The experiment related to MII oocyte collection was performed according to the previously described methods with minor modification (Kang et al., 2014; Kim and Lee, 2019; So et al., 2020). Female mice were super-ovulated with 5 international units (IU) pregnant mare’s serum gonadotropin (PMSG), followed by 5 IU human chorionic gonadotropin (hCG) 48 h later. MII oocytes were collected 16 h after hCG injection. The denuded oocytes by 0.1% hyaluronidase solution (Sigma) were kept in KSOM (Merck) at 37℃ in a humidified incubator with 5% CO2.
Oocytes were incubated in an activation medium supplemented with 10 mM SrCl2 (Sigma) in the presence or absence of 5 ug/mL cytochalasin B (CB, Sigma) for six hours at 37℃ in a humidified incubator with 5% CO2. After activation, the oocytes were examined for the formation of pronucleus (PN). Activated oocytes were cultured for four to five days to the blastocysts in KSOM (Merck) at 37℃ in a humidified incubator with 5% CO2 (Lee and Kang, 2021).
2-cell embryos are placed between the electrodes of a 100 ?m gap electrode chamber (BLS Ltd.) containing 0.28 M Mannitol (Sigma), 0.1 mM magnesium sulfate hydrate (Sigma), 50 ?M calcium chloride (Sigma), and 0.3% bovine serum albumin (Sigma) and fused by Electro Cell Manipulator ECM 2001 (BTX). An initial electrical field of 2V is applied to the embryos followed by one peak pulse of 50V for 35 ?s. After fusion, the embryos were transferred to KSOM and observed for fusion.
Denuded blastocysts were placed onto mitomycin C (Sigma)-treated mouse embryonic fibroblast (MEF) feeder in mESC medium: KODMEM (Gibco) containing 20% KOSR (Gibco), 1 mM L-glutamine (Gibco), 100 units/mL penicillin (Hyclone), 100 ?g/mL streptomycin (Hyclone), 100 ?M β-mercaptoethanol (Sigma), 100 ?M nonessential, and 1,000 units/m LIF (Stemgent) under 5% CO2 at 37℃ in a humidified incubator. The cell outgrowth was replaced on the new MEF feeder. mESCs were passaged every 3 to 4 days for further experiments.
Cells were fixed with 70% ethanol at 4℃ for 15 min and stained with 50 ?g/mL propidium iodide (Sigma) solution containing 100 ?g/mL RNase A at 4℃ for 40 min. The cell cycle was measured by a FACS Calibur (BD Biosciences) and the data were analyzed by FlowJo v10.5.3 (FlowJo LLC).
KaryoMAX Colcemide (Gibco) with 150 ng/mL concentration was applied to the cultured ESCs for 1.5 h at 37℃. The treated cells were detached by trypsin/EDTA. The cells treated with 0.075 M KCL hypotonic solution for 30 min were fixed with methanol: acetic acid (3:1 v/v) and dropped onto a slide. The slides were mounted in Prolong Diamond Antifade Mountant with DAPI (Invitrogen).
Data were analyzed with Fisher’s exact test for two groups and ANOVA with Tukey analysis for multiple comparisons. Statistical analyses were performed using GraphPad Prism (version 6). Data are expressed as mean ± standard error of the mean (S.E.M), and
To produce diploid parthenogenic embryos by activation, MII oocytes were activated with CB, resulting in that the zygotes could show two pronuclei (2 PNs) and no extrusion of the 2nd PB after activation (Bałakier and Tarkowski, 1976) (SrCl2+CB group in Fig. 1A). While, for the generation of haploid embryos, MII oocytes were activated without CB, and the zygote could show one pronucleus (1 PN) and the extrusion of the 2nd PB (SrCl2-CB group in Fig. 1A). Some haploid blastomeres on the 2-cell stage were performed electronic fusion to induce diploidy (SrCl2-CB with 2-cell fusion group in Fig. 1A).
After activation, the PN formation rate in the SrCl2+CB and SrCl2-CB group was comparable (100% vs. 97%) (Fig. 1B). Both groups showed the embryo development to blastocyst (Fig. 1C). The embryo development from 2-cell to morula stage was similar between the SrCl2+CB and SrCl2-CB group (100% vs. 96% for 2-cell; 100% vs. 89% for 4-cell; 100% vs. 91% for morula). However, the blastocyst rate was significantly lower in an SrCl2-CB group than in the SrCl2+CB group (37% vs. 100%) (Fig. 1B).
When the 2-cell embryos in the SrCl2-CB group were fused electronically, the embryos were changed their morphology to one cell, cleaved again on the next day, and developed into blastocysts (Fig. 1C). The embryo development from 2-cell to morula stage was 86% for 2-cell, 94% for 4-cell, and 91% for morula in SrCl2-CB with 2-cell fusion group, which was comparable to SrCl2+CB and SrCl2-CB group (Fig. 1B). However, the blastocyst rate was significantly higher in the SrCl2-CB with 2-cell fusion group than in the SrCl2-CB group (55% vs. 37%), which was significantly lower than in the SrCl2+CB group (100%) (Fig. 1B).
During the embryo development, the blastocysts in the SrCl2+CB group were formed on day 4 and hatched on day 5 (Fig. 1C). While, in the SrCl2-CB group, the blastocysts were started to appear on day 5 and the quality of blastocyst was worse compared to the SrCl2+CB group (Fig. 1C). Based on this result, we examined what embryo stage was delayed during the embryo culture in the SrCl2-CB group. We compared the distribution of 2-cell at 32 hours, 4-cells at 56 hours, morula at 80 hours, and blastocyst at 120 and 128 hours in the cultured embryos of SrCl2+CB group, SrCl2-CB group, and SrCl2-CB with 2-cell fusion group (Fig. 1D). There was no significant difference in 2-cell distribution at 32 hours between the SrCl2+CB group and SrCl2-CB group. However, the distribution of 4-cells at 56 hours, morula at 80 hours, and blastocyst at 120 and 128 hours was significantly lower in the SrCl2-CB group compared to the SrCl2+CB group. In SrCl2-CB with 2-cell fusion group, the distribution of morula at 80 hours, and blastocyst at 120 and 128 hours was significantly higher than SrCl2-CB group but lower than SrCl2+CB group. Based on results, the SrCl2-CB group showed developmental delay from 4-cell to blastocyst stage while SrCl2-CB with 2-cell fusion group displayed the improved development delay.
ESCs were established from the blastocysts in each group and the cell cycle analysis was performed (Fig. 2A and 2B). The mouse fibroblasts were used as a diploid control. ESCs in SrCl2+CB and SrCl2-CB with 2-cell fusion group were confirmed the diploid (Fig. 2B). While ESCs in the SrCl2-CB group displayed haploid. We confirmed the haploid in the SrCl2-CB group and diploid in SrCl2-CB with the 2-cell fusion group by karyotyping (Fig. 2C). The rate of ESC establishment from the blastocysts was significantly lower in the SrCl2-CB group compared to SrCl2+CB and SrCl2-CB with 2-cell fusion group (5.3% vs. 78.3% and 51.5%) (Fig. 2D). The rate in SrCl2-CB with 2-cell fusion group was lower than SrCl2+CB group but was not a significant difference. When the rate of ESC establishment was calculated based on the number of MII oocytes, the SrCl2-CB group showed a significantly lower rate than SrCl2+CB and SrCl2-CB with 2-cell fusion group (1.4% vs. 78.3% and 20%) (Fig. 2D). The rate in SrCl2-CB with the 2-cell fusion group was significantly lower than in the SrCl2+CB group. These results suggested that electronic fusion could increase the rate of homozygous ESC establishment.
The present study demonstrated whether the electronic fusion of haploid embryos could induce the increase of homozygous ESCs establishment. Electronic fusion of haploid embryos induced significantly higher blastocyst generation and ESC establishment compared to non-fused ones.
In mice, parthenogenetic embryos usually maintained haploidy at the blastocyst stage (Tarkowski et al., 1970; Kaufman et al., 1983), however, post-implantation haploid embryos tended to become diploid
Previous studies demonstrated that haploid embryos showed a lower developmental rate in preimplantation embryos than the diploid embryos (Witkowska, 1973; Kaufman and Sachs, 1976; Latham et al., 2002), which was also observed in our study. Further, we demonstrated that the developmental delay of haploid embryos occurred in 4-cell to the blastocyst stage. Electronic fusion was performed in the haploid 2-cell, resulting in the blastocyst rate being significantly increased compared to non-fused embryos. Further, the rate of ESCs establishment was higher in fused embryos than non-fused one, suggesting more homozygous ESCs could be generated due to the higher efficiency of blastocyst and ESC generation.
Electronic fusion of 2-cell blastomeres is often used for tetraploid complementation (Gertsenstein, 2015). When we applied the same method in haploid 2-cells, the embryo development of 2-cell to morula was comparable to the non-fused haploid embryo, further the blastocyst rate was significantly higher, resulting in the electronic fusion did not damage the embryo development and quality in haploid embryos.
There is another method to produce parthenogenic haploid embryos, which involves removing the male PN from the fertilized zygote (Bai et al., 2016). However, this method requires complex processes, such as fertilization and removing PN by manipulation, and is hard to apply in humans due to the ethical issue of destroying the fertilized embryos. Therefore, the activation of mature oocytes could be a simple and efficient method to produce a parthenogenetic haploid embryo. Further, the electronic fusion of the haploid embryo could induce a higher rate of blastocyst development, which could lead to higher efficiency for the generation of ESCs with homozygosity.
Haploidization of the diploid somatic genome was tried using mouse oocytes (Tateno et al., 2003; Lee et al., 2022). The method described in the current article could be applied in somatic haploid embryos to improve the establishment of somatic homozygous ESCs.
Haploid embryos showed a lower blastocyst rate and the developmental delay from 4-cell to blastocyst stage compared to diploid embryos. When the electronic fusion was performed in haploid embryos, the developmental delay was improved. The rates of blastocyst development and ESC establishment were significantly increased compared to the non-fused group. Based on the result, we concluded that the electronic fusion of haploid embryos could be an efficient method to generate homozygous ESCs.
None.
Conceptualization, Y.L., E.K.; data curation, B.K., Y.L.; formal analysis, B.K., S.S., J.C., Y.L.; funding acquisition, Y.L., E.K.; investigation, B.K., S.S., J.C., Y.L.; methodology, B.K., S.S., J.C., Y.L.; project administration, Y.L., E.K.; resources, B.K., Y.L.; software, B.K., J.C., Y.L.; supervision, Y.L., E.K.; validation, B.K., S.S., J.C., Y.L.; visualization, B.K., Y.L.; writing - original draft, B.K.; writing - review & editing, Y.L., E.K.
This work has been supported by the National Research Foundation of Korea (grant nos. NRF-2018R1A2B3001244 and NRF-2021R1I1A1A01049705).
The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC No.210035) of CHA University.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
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