Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Lee, Joohyeong; Kim, Eunhye; Hwang, Seon‐Ung; Cai, Lei; Kim, Mirae; Choi, Hyerin; Oh, Dongjin; Lee, Eunsong; Hyun, Sang Hwan

doi:10.3390/ani11041034

Cited by 7 publications

(8 citation statements)

References 43 publications

(10 reference statements)

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“…After IVM, the MII-stage oocytes were selected to conduct PA as described by Lee et al ( Lee et al, 2021 ). The oocytes were washed twice in the activation solution containing 280 mM mannitol, 0.01 mM CaCl 2, and 0.05 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines

Lee

Cai²,

Kim³

et al. 2022

Front. Cell Dev. Biol.

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Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly (p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.

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Section: Methodsmentioning

confidence: 99%

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines

Lee

Cai²,

Kim³

et al. 2022

Front. Cell Dev. Biol.

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“…The IVM of oocytes was performed in the same manner as described by Lee et al ( Lee et al, 2021 ). The ovaries of prepubertal gilts obtained from a local abattoir were transported to the laboratory at 34°C–37°C.…”

Section: Methodsmentioning

confidence: 99%

“…The PA of oocytes for FUTP and CBTP methods were performed as previously described by Lee et al ( Lee et al, 2021 ). Only oocytes from which the PB1 was released after IVM were selected for PA.…”

Section: Methodsmentioning

confidence: 99%

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs

Lee,

Cai,

Kim

et al. 2023

Front. Cell Dev. Biol.

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Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.

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“…NT and PA were performed as previously described in Lee et al [18]. For NT, green fluorescent protein (GFP)-inserted porcine fetal fibroblasts were used as NT donor nuclei.…”

Section: Pa and Ntmentioning

confidence: 99%

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

et al. 2023

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The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

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Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Cited by 7 publications

References 43 publications

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

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