Koki Takebayashi, Manita Wittayarat, Maki Hirata, Qingyi Lin, Zhao Namula, Nanaka Torigoe, Bin Liu, Megumi Nagahara, Aya Nakai, Takeshige Otoi and Fuminori Tanihara : Optimization of embryonic stage for aggregation to generate chimeric pigs using gene-edited blastomeres., In Vitro Cellular & Developmental Biology. Animal, 2024.
Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Zhao Namula, Maki Hirata, Fuminori Tanihara, Megumi Nagahara and Takeshige Otoi : Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system., The Journal of Reproduction and Development, 2024.
(要約)
CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.
Megumi Nagahara, Zhao Namula, Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Fuminori Tanihara, Takeshige Otoi and Maki Hirata : Effects of ergothioneine supplementation on meiotic competence and porcine oocyte development., Veterinary World, Vol.17, No.8, 1748-1752, 2024.
(要約)
Supplementing with EGT during IVM leads to better oocyte maturation, quality, and embryonic development due to decreased DNA fragmentation. The present study failed to elucidate the mechanism of DNA fragmentation reduction by EGT. More research needs to be conducted to explore the antioxidant mechanism of EGT.
Bin Liu, Manita Wittayarat, Koki Takebayashi, Qingyi Lin, Nanaka Torigoe, Zhao Namula, Maki Hirata, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Effects of centrifugation treatment before electroporation on gene editing in pig embryos., In Vitro Cellular & Developmental Biology. Animal, 2024.
(要約)
Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.
Thi Suong Nguyen, Masayasu Taniguchi, Tetsushi Ono, Mitsuhiro Takagi, Qingyi Lin, Nanaka Torigoe, Bin Liu, Zhao Namula, Megumi Nagahara and Takeshige Otoi : Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method., Reproduction in Domestic Animals = Zuchthygiene, Vol.59, No.6, 2024.
(要約)
We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.
(キーワード)
Animals / Male / Caffeine / Spermatozoa / Fertilization in Vitro / Cryopreservation / Semen Preservation / Female / Sperm Motility / Swine / Embryonic Development / Oocytes / Blastocyst
Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Zhao Namula, Maki Hirata, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes., In Vitro Cellular & Developmental Biology. Animal, 2024.
(要約)
The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.
Thanh-Van Nguyen, Kim Lanh Thi Do, Qingyi Lin, Megumi Nagahara, Zhao Namula, Manita Wittayarat, Maki Hirata, Takeshige Otoi and Fuminori Tanihara : Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes., In Vitro Cellular & Developmental Biology. Animal, 2024.
(要約)
Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.
Thanh-Van Nguyen, Koki Takebayashi, Kim Lanh Thi Do, Zhao Namula, Manita Wittayarat, Megumi Nagahara, Maki Hirata, Takeshige Otoi and Fuminori Tanihara : Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs., In Vitro Cellular & Developmental Biology. Animal, 2024.
(要約)
The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.
Supitcha Kaewma, Zhao Namula, Thi Suong Nguyen, Qingyi Lin, Nanaka Torigoe, Bin Liu, Megumi Nagahara, Masahiro Nii, Masayasu Taniguchi and Takeshige Otoi : Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen., Acta Veterinaria Hungarica, Vol.71, No.3-4, 219-222, 2024.
(要約)
This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.
T Suong Nguyen, Ayane Edo, Megumi Nagahara, Takeshige Otoi, Masayasu Taniguchi and Mitsuhiro Takagi : Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device., Animal Reproduction Science, Vol.260, No.260, 2024.
(要約)
This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2-4 cell stage. The intracellular ROS concentrations were investigated using 2', 7'-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.
Nanaka Torigoe, Megumi Nagahara, Thi Suong Nguyen, Qingyi Lin, Koki Takebayashi, Bin Liu, Mutsumi Aihara, Masayasu Taniguchi and Takeshige Otoi : Development of porcine embryos cultured in media irradiated with ultraviolet-C., Reproduction in Domestic Animals = Zuchthygiene, Vol.59, No.1, e14520, 2024.
(要約)
Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.
Shogo Hashimoto, Masayasu Taniguchi, Ayane Edo, Tetsushi Ono, Tetty Barunawati Siagian, Hiroaki Sekine, Megumi Nagahara, Takeshige Otoi and Mitsuhiro Takagi : Impact of redox status of donor cows before superovulation treatment on in vivo embryo production., Archives Animal Breeding, Vol.66, No.4, 433-437, 2023.
Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Zhao Namula, Maki Hirata, Fuminori Tanihara, Megumi Nagahara and Takeshige Otoi : Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos., Animal Science Journal, Vol.94, No.1, e13878, 2023.
(要約)
The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.
Hisayoshi Omori, Junko Chikamoto, Megumi Nagahara, Maki Hirata and Takeshige Otoi : Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system., Toxicology In Vitro, Vol.93, 2023.
(要約)
Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.
Chommanart Thongkittidilok, Maki Hirata, Qingyi Lin, Nanaka Torigoe, Bin Liu, Yoko Sato, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Mosaic TP53 Mutation on Tumour Development in Pigs: A Case Study., Veterinary Medicine International, Vol.2023, 7000858, 2023.
(要約)
mutations with truncated amino acids may be related to tumour formation.
Fuminori Tanihara, Maki Hirata, Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Hiromasa Hara, Megumi Nagahara and Takeshige Otoi : GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system., Molecular Biology Reports, Vol.50, No.6, 5049-5057, 2023.
(要約)
Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.
Megumi Nagahara, Zhao Namula, Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Mutsumi Aihara, Masahiro Nii and Takeshige Otoi : Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation., Reproduction in Domestic Animals = Zuchthygiene, 2023.
(要約)
It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 μM curcumin under UV-C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 μM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p < .05). In the second experiment, semen was preserved with or without 25 μM curcumin with UV-C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p < .05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long-duration irradiation with 228 nm UV-C. Curcumin supplementation improves the quality of UV-C irradiated spermatozoa during semen preservation.
Thanh-Van Nguyen, Kim Lanh Thi Do, Zhao Namula, Qingyi Lin, Nanaka Torigoe, Megumi Nagahara, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Development and Genome Mutation of Bovine Zygotes Vitrified Before and After Genome Editing via Electroporation, Cryo Letters, Vol.44, No.2, 118-122, 2023.
Qingyi Lin, Mutsumi Aihara, Akihiro Shirai, Ami Tanaka, Koki Takebayashi, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Takeo Minamikawa and Takeshige Otoi : Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm, Theriogenology, Vol.197, 252-258, 2023.
(要約)
It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV-C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Nanaka Torigoe, Bin Liu, Maki Hirata, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene., Animal Science Journal, Vol.94, No.1, e13842, 2023.
(要約)
Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.
(キーワード)
Male / Animals / Swine / Gene Editing / Zygote / CRISPR-Cas Systems / Semen / Electroporation / RNA, Guide, CRISPR-Cas Systems
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Maki Hirata, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Kim Lanh Thi Do, Fuminori Tanihara and Takeshige Otoi : Gene editing in porcine embryos using a combination of electroporation and transfection methods., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.10, 1136-1142, 2022.
(要約)
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.
(キーワード)
Animals / CRISPR-Associated Protein 9 / CRISPR-Cas Systems / Electroporation / Gene Editing / Swine / Transfection / Zygote
Megumi Shimazaki, Manita Wittayarat, Rentsenkhand Sambuu, Asami Sugita, Masaki Kawaguchi, Maki Hirata, Fuminori Tanihara, Mitsuhiro Takagi, Masayasu Taniguchi, Takeshige Otoi and Yoko Sato : Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.9, 999-1006, 2022.
(要約)
The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2-3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.
Praopilas Phakdeedindan, Manita Wittayarat, Theerawat Tharasanit, Mongkol Techakumphu, Megumi Shimazaki, Rentsenkhand Sambuu, Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi and Yoko Sato : Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.3, 304-313, 2021.
(要約)
Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.
(キーワード)
Acetylation / Animals / Cattle / Cattle Diseases / DNA Methylation / Infertility, Male / Male / Spermatogenesis / Testis
In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle-yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle-yak spermatogenesis. Testicular tissues from yaks (1-3 years old) and hybrids (F1-F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle-yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle-yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.
Megumi Shimazaki, Urasoko Saki, Tanaka Masako, Sato Yoko, Fuminori Tanihara, Maki Hirata, Taniguchi Masayasu, Takagi Mitsuhiro and Takeshige Otoi : Effects of Orvus Es Paste (OEP) on the viability of bull spermatozoa after double freezing and thawing., The International Journal of Applied Research in Veterinary Medicine, Vol.16, 32-38, 2018.
Megumi Shimazaki, Rentsenkhand Sambuu, Yoko Sato, Kim Lanh Thi Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING., Cryo Letters, Vol.36, No.4, 264-269, 2015.
(要約)
The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. Semen samples were frozen and thawed in semen freezing extender supplemented with 0 %, 0.375 %, 0.75 % or 1.5 % OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. The addition of 0.75 % OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3h of incubation. Our findings indicate that the addition of 0.75 % OEP is effective for the preservation of yak spermatozoa.
B Liu, Takeshige Otoi, TAKEBAYASHI Kohki, Wittayarat Manita, Maki Hirata, Q. Lin, N. Torigoe, Megumi Nagahara, Namula Zhao and Fuminori Tanihara : Trial to generate chimeric pigs with high-frequency renal tumors via aggregation of gene-edited blastomeres., 27th Annual ESDAR Conference, Sep. 2024.
2.
Li Qingyi, K. Takebayashi, N. Torigoe, Liu Bin, Maki Hirata, Fuminori Tanihara, Megumi Nagahara and Takeshige Otoi : Genome editing of porcine zygotes through the lipofection of a CRISPR/Cas9 system with two guide RNAs., The 50th Conference of the International Embryo Technology Society, Jan. 2024.
3.
Suong T. Nguyen, Masayasu Taniguchi, S. Kaewma, Megumi Nagahara, Mitsuhiro Takagi and Takeshige Otoi : Quality and fertilization of frozenthawed porcine spermatozoa separated using migration sedimentation., The 50th Conference of the International Embryo Technology Society, Jan. 2024.
4.
N. Torigoe, Mutsumi Aihara, Q. Lin, K. Takebayashi, B. Liu, Megumi Nagahara and Takeshige Otoi : Development of porcine embryos cultured in media irradiated with ultraviolet at 228 and 260 nm wavelength., The 50th Conference of the International Embryo Technology Society, Jan. 2024.
? 斌, 長原 恵, Namula Zhao, 林 青怡, 竹林 滉生, 鳥越 菜々花, 音井 威重 : Effect of porcine follicle fluid with the different oxidation stress indices on the meiotic competence and DNA integrity of porcine oocytes, 117, 2024年9月.
3.
Lin Qingyi, Takebayashi Koki, Torigoe Nanaka, Liu Bin, Maki Hirata, Megumi Nagahara and Takeshige Otoi : Efficient gene editing of pig embryos by combining electroporation and lipofection methods depends on gRNA sequence., 第117回日本繁殖生物学会, Sep. 2024.
佐藤 陽子, 長原 恵, R Ogasawara, Y Obatake, K Kawanishi, H Obatake, K Shibata, A Kinebuchi, Y Higashihara, K Sugaya, R Sambuu, Y Tanighuchi, 音井 威重 : ヤクー牛雑種の雄性不稔に関わる精巣上体細胞サイズの検討, 第46回日本分子生物学会年会, 2023年12月.