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.
(Summary)
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.
(Summary)
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.
(Summary)
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.
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.
(Summary)
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.
(Summary)
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.
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.
(Summary)
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.
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.
(Summary)
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.
(Summary)
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.
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.
Fuminori Tanihara, Maki Hirata, Zhao Namula, Kim Lanh Thi Do, Naoaki Yoshimura, Qingyi Lin, Koki Takebayashi, Tetsushi Sakuma, Takashi Yamamoto and Takeshige Otoi : Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN, Frontiers in Cell and Developmental Biology, Vol.11, 2023.
(Summary)
electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.
Anh Quynh Le, Manita Wittayarat, Zhao Namula, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Kim Lanh Thi Do and Takeshige Otoi : Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods., Veterinary World, Vol.15, No.9, 2210-2216, 2022.
(Summary)
The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts.
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.
(Summary)
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.
(Keyword)
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.
(Summary)
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.
We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of β-cells in human disease such as type 1 diabetes.
Zhao Namula, Manita Wittayarat, Kim Lanh Thi Do, Thanh Nguyen Van, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system, Veterinary and Animal Science, Vol.16, 2022.
Zhao Namula, Anh Quynh Le, Manita Wittayarat, Qingyi Lin, Koki Takebayashi, Maki Hirata, Kim Lanh Thi Do, Fuminori Tanihara and Takeshige Otoi : Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection., Veterinary World, Vol.15, No.2, 496-501, 2022.
(Summary)
These results indicate that although a combination of MI and EP does not improve the mutation efficiency or biallelic mutation for triple-gene knockout, MI treatment before EP is better to reduce mortality in porcine zygotic gene editing through a combination of MI and EP.
Qingyi Lin, Anh Quynh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Chommanart Thongkittidilok, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.5, 556-563, 2022.
(Summary)
-W [Cell-W] and cell suspension and preservation solution, Cellstor
(Keyword)
Animals / Blastocyst / Culture Media / Curcumin / Embryo, Mammalian / Fertilization in Vitro / Swine / Temperature
Qingyi Lin, Anh Quynh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : Short-term preservation of porcine zygotes at ambient temperature using a chemically defined medium., Animal Science Journal, Vol.93, No.1, 2022.
(Summary)
We aimed to develop a simple method for the short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell-W and Cell-S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell-W solution for 24-72 h at 25°C. The Cell-W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short-term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis.
(Keyword)
Animals / Blastocyst / Culture Media / Fertilization in Vitro / Swine / Temperature / Zygote
Chommanart Thongkittidilok, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Lanh Thi Kim Do, Zhao Namula, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.3, 314-320, 2021.
(Summary)
The oocyte maturation process requires a high supply of energy, which generates reactive oxygen species (ROS), adversely affecting oocyte and embryo development. Balancing ROS by antioxidant supplementation is essential for maintaining oocyte maturation and embryonic quality in vitro. This study aimed to evaluate the impact of four antioxidants: β-mercaptoethanol (β-ME), chlorogenic acid (CGA), curcumin and sericin, when applied individually or in combinations, during oocyte maturation on development of porcine oocytes. Cumulus-oocyte complexes were collected, cultured in maturation medium supplemented with antioxidants for 44 hr and subsequently subjected to in vitro fertilization (IVF) and culture for 7 days. Combining all four (β-ME + CGA + curcumin + sericin) or three (β-ME + CGA + curcumin) antioxidants increased blastocyst formation rates. However, sericin supplementation alone, or in combination with β-ME or CGA, failed to improve blastocyst formation rates. The total cell numbers of blastocysts from the group supplemented with three antioxidants (β-ME + CGA + curcumin) were significantly higher than those from the other groups, except for the curcumin-supplement group. There were no differences in the maturation rates and proportions of oocytes with fragmented DNA between the antioxidant-supplemented and the non-supplemented control groups. In conclusion, supplementation with three antioxidants (β-ME + CGA + curcumin) during the maturation culture enhanced blastocyst formation and improved blastocyst quality.
(Keyword)
Animals / Antioxidants / Blastocyst / Dietary Supplements / Embryonic Development / Fertilization in Vitro / In Vitro Oocyte Maturation Techniques / Oocytes / Swine
Fuminori Tanihara, Maki Hirata, Thi Nhien Nguyen, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis., International Journal of Molecular Sciences, Vol.22, No.5, 2249, 2021.
(Summary)
triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Le Anh, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Fuminori Tanihara and Takeshige Otoi : Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos., Animals : An Open Access Journal from MDPI, Vol.11, No.2, 2021.
(Summary)
Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.
Anh Quynh Le, Fuminori Tanihara, Manita Wittayarat, Zhao Namula, Yoko Sato, Qingyi Lin, Koki Takebayashi, Maki Hirata and Takeshige Otoi : Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages., BMC Research Notes, Vol.14, No.1, 7, 2021.
(Summary)
Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos.
Kim Thi Lanh Do, Manita Wittayarat, Yoko Sato, Kaywalee Chatdarong, Theerawat Tharasanit, Mongkol Techakumphu, Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : Comparison of blastocyst development between cat-cow and cat-pig interspecies somatic cell nuclear transfer embryos under the treatment of Trichostatin A., Biology bulletin of the Russian Academy of Sciences, Vol.48, 107-117, 2021.
Nguyen Nhien Thi, Wittayarat Manita, Zhao Namula, Sato Yoko, Anh Le Quynh, Lin Qingyi, Takebayashi Koki, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Chlorogenic acid and insulin-transferrin-selenium supplementation during in vitro maturation enhances the developmental competence of interspecies chimera blastocysts following cell injection., Journal of Applied Animal Research, Vol.49, No.1, 486-491, 2021.
Manita Wittayarat, Maki Hirata, Zhao Namula, Yoko Sato, T Nhien Nguyen, A Quynh Le, Qingyi Lin, Koki Takebayashi, Fuminori Tanihara and Takeshige Otoi : Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides., Animal Science Journal, Vol.92, No.1, e13534, 2021.
(Summary)
This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.
Fuminori Tanihara, Maki Hirata, Thi Nhien Nguyen, Le Anh Quynh, Manita Wittayarat, Mokhamad Fahrudin, Takayuki Hirano and Takeshige Otoi : Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes., Animal Biotechnology, Vol.32, 147-154, 2021.
Maki Hirata, Manita Wittayarat, Fuminori Tanihara, Yoko Sato, Zhao Namula, Anh Quynh Le, Qingyi Lin, Koki Takebayashi and Takeshige Otoi : One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs., In Vitro Cellular & Developmental Biology. Animal, Vol.56, No.8, 614-621, 2020.
(Summary)
In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.
Maki Hirata, Manita Wittayarat, Zhao Namula, Anh Quynh Le, Qingyi Lin, Thi Nhien Nguyen, Koki Takebayashi, Yoko Sato, Fuminori Tanihara and Takeshige Otoi : Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation., Molecular Biology Reports, Vol.47, No.7, 5073-5079, 2020.
(Summary)
The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.
Fuminori Tanihara, Maki Hirata, Nhien Nguyen Thi, Le Anh Quynh, Takayuki Hirano and Takeshige Otoi : Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics., Molecular Reproduction and Development, Vol.87, No.4, 471-481, 2020.
(Summary)
Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.
T N Nguyen, Maki Hirata, Fuminori Tanihara, Y Sato, Z Namula, Le Trong Quang, M Wittayarat, M Fahrudin and Takeshige Otoi : In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification., Cryo Letters, Vol.41, No.2, 86-91, 2020.
(Summary)
Our results suggest that the removal of the ZP does not cause detrimental effects to the development of vitrified-warmed zygotes.
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.
Namula Zhao, Yoko Sato, Manita Wittayarat, Quynh Le Anh, Thi Nhien Nguyen, Qingyi Lin, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Curcumin supplementation in maturation medium improves the maturation, fertilisation, and developmental competence of porcine oocytes., Acta Veterinaria Hungarica, Vol.68, No.3, 298-304, 2020.
(Summary)
This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.
(Keyword)
Animals / Blastocyst / Cell Proliferation / Curcumin / DNA Fragmentation / Dose-Response Relationship, Drug / Embryo Culture Techniques / Hydrogen Peroxide / In Vitro Oocyte Maturation Techniques / Oocytes / Oxidative Stress / Sus scrofa
A Quynh Le, Maki Hirata, T Nhien Nguyen, Koki Takebayashi, Manita Wittayarat, Yoko Sato, Zhao Namula, Masahiro Nii, Fuminori Tanihara and Takeshige Otoi : Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes., Animal Science Journal, Vol.91, No.1, e13386, 2020.
(Summary)
This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.
Maki Hirata, Manita Wittayarat, Takayuki Hirano, Thi Nhien Nguyen, Le Anh Quynh, Zhao Namula, Mokhamad Fahrudin, Fuminori Tanihara and Takeshige Otoi : The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) pol genes in porcine embryos., Animals : An Open Access Journal from MDPI, Vol.9, No.9, E593, 2019.
Namula Zhao, Manita Wittayarat, Maki Hirata, Hirano Takayuki, Nguyen Thi Nhien, Le Anh Quynh, Fahrudin Mokhamad, Fuminori Tanihara and Takeshige Otoi : Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes, In Vitro Cellular & Developmental Biology. Animal, Vol.55, No.8, 598-603, 2019.
(Summary)
The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P<0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P<0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.
Fuminori Tanihara, Maki Hirata, Morikawa Shigeki, Thi Nhien Nguyen, Le Anh Quynh, Hirano Takayuki, Fukumi Yoshiyuki, Abe Toshiaki and Takeshige Otoi : The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts, The Journal of Reproduction and Development, Vol.65, No.5, 475-479, 2019.
(Summary)
The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation.
Fuminori Tanihara, Maki Hirata, Satoru Iizuka, Shinya Sairiki, Masahiro Nii, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano and Takeshige Otoi : Relationship among ovarian follicular status, developmental competence of oocytes, and anti-M llerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts., Animal Science Journal, Vol.90, No.6, 712-718, 2019.
(Summary)
The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti-M llerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross-sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.
Nhien Nguyen Thi, Maki Hirata, Fuminori Tanihara, Takayuki Hirano, Quynh Anh Le, Masahiro Nii and Takeshige Otoi : Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid., Reproduction in Domestic Animals = Zuchthygiene, Vol.54, No.5, 750-755, 2019.
(Summary)
The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25 C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25 C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25 C for 24 hr was evaluated, more zygotes stored with 50 M CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 M CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 M CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.
Zhao Namula, Fuminori Tanihara, Manita Wittayarat, Maki Hirata, Nhien Thi Nguyen, Takayuki Hirano, Quynh Anh Le, Masahiro Nii and Takeshige Otoi : Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa., Acta Veterinaria Hungarica, Vol.67, No.1, 106-114, 2019.
(Summary)
Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 M) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 M of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 M of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 M did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 M Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 M Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing.
Maki Hirata, Fuminori Tanihara, Manita Wittayarat, Takayuki Hirano, Nhien Thi Nguyen, Quynh Anh Le, Zhao Namula, Masahiro Nii and Takeshige Otoi : Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes., In Vitro Cellular & Developmental Biology. Animal, Vol.55, No.4, 237-242, 2019.
(Summary)
The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh LE, Takayuki Hirano and Takeshige Otoi : Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos., The Journal of Reproduction and Development, Vol.65, No.3, 209-214, 2019.
(Summary)
Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/ l of Cas9 protein and guide RNA (gRNA), targeting the -1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/ l each (20 ng/ l group) or 100 ng/ l each (100 ng/ l group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/ l group was significantly higher (P < 0.05) than that in the 20 ng/ l group. Although no blastocysts from the 20 ng/ l group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/ l group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.
Fuminori Tanihara, Maki Hirata, Nhien T. Nguyen, Quynh A. Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation., Animal Science Journal, Vol.90, No.1, 55-61, 2018.
(Summary)
Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation., PLoS ONE, Vol.13, No.10, e0206360, 2018.
(Summary)
TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.
Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi, Mitsuhiro Takagi, Tsukasa Terazono and Takeshige Otoi : Follicular development of canine ovaries stimulated by a combination treatment of eCG and hCG., Veterinary Medicine and Science, 2018.
(Summary)
Ovarian follicular dynamics is not well known in dogs. Imaging of ovaries is technically difficult; however, ovaries clamped at a subcutaneous site can more easily be monitored using ultrasound imaging. This study investigated the follicular development of canine ovaries stimulated by hormone treatment using ultrasound imaging of the ovaries clamped at a subcutaneous site. Oestrus was induced using subcutaneous administration of 500 IU equine chorionic gonadotropin (eCG) and 1000 IU human chorionic gonadotropin (hCG) (eCG/hCG). Five bitches were given 1000 IU hCG 11 days after eCG/hCG administration. Examinations with ovarian ultrasonography using a 7.5-MHz sector transducer, vaginal cytology, and assays of serum oestrogen and progesterone were performed daily until 20 days after eCG/hCG administration. Serosanguineous vaginal discharges and vaginal cytology of two of the bitches were observed. New follicular growth (>1.0 mm in diameter) was observed in all bitches from 2 to 8 days after eCG/hCG administration. The mean diameter of follicles and maximum numbers of follicles per ovary ranged from 2.8 to 5.5 mm and 4 to 16, respectively. The elevation in oestrogen concentrations after eCG/hCG administration was observed in all bitches, and elevation in progesterone concentration (>2 ng mL ) was observed in three bitches. However, no follicles ovulated until 9 days after hCG administration. In conclusion, although the number of examined bitches were limited, follicular growth in ovaries clamped at a subcutaneous site can be monitored using ultrasound imaging. Ovarian ultrasonography showed that eCG/hCG administration induced new follicular growth and hCG administration induced increases in oestrogen concentrations but not ovulation by hCG administration.
Maki Hirata, Fuminori Tanihara, Taniguchi Masayasu, Takagi Mitsuhiro, Terazono Tsukasa and Takeshige Otoi : Viability of canine ovaries autografted to different peripheral sites, The International Journal of Applied Research in Veterinary Medicine, Vol.16, No.2, 140-148, 2018.
Zhao Namula, Maki Hirata, Manita Wittayarat, Fuminori Tanihara, Nhien Nguyen Thi, Takayuki Hirano, Masahiro Nii and Takeshige Otoi : Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm., Reproduction in Domestic Animals = Zuchthygiene, 2018.
(Summary)
Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity.
Tetsushi Ono, Mitsuhiro Takagi, Chiho Kawashima, B Missaka P Wijayagunawardane, M Peter L A Vos, Masayasu Taniguchi, Fuminori Tanihara and Takeshige Otoi : Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles., Frontiers in Veterinary Science, Vol.5, 2018.
(Summary)
= 0.07) for group hCG-1 compared to group hCG-C on day 5. The preliminary findings of this study suggest that multiple smaller doses of hCG might be equally effective as a single large dose of hCG in modulating ovarian follicular development in dairy cows with COFs.
Thanh-Van Nguyen, Manita Wittayarat, Kim Lanh Thi Do, Van Thanh Nguyen, Masahiro Nii, Zhao Namula, Toshiki Kunihara, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization., Animal Science Journal, Vol.89, No.8, 1207-1213, 2018.
(Summary)
Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation-treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation-treated embryos.
Fuminori Tanihara, Maki Hirata, Thi Nguyen Nhien, Takayuki Hirano, Toshiki Kunihara and Takeshige Otoi : Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation., The Journal of Veterinary Medical Science, Vol.80, No.6, 1007-1011, 2018.
(Summary)
The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.
V T Nguyen, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Katsutoshi Nishio, T Do L Kim, V T Nguyen, M Nii and Takeshige Otoi : Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures., Cryo Letters, Vol.39, No.2, 131-136, 2018.
(Summary)
Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.
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.
Katsutoshi Nishio, Fuminori Tanihara, T-V Nguyen, Toshiki Kunihara, M Nii, Maki Hirata, Tatsuya Takemoto and Takeshige Otoi : Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.53, No.2, 313-318, 2017.
(Summary)
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
T-V Nguyen, Fuminori Tanihara, Ltk Do, Y Sato, M Taniguchi, M Takagi, T Nguyen Van and Takeshige Otoi : Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes., Reproduction in Domestic Animals = Zuchthygiene, Vol.52, No.6, 969-975, 2017.
(Summary)
Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H2 O2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2 O2 to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.
Katsutoshi Nishio, Mado Yamazaki, Masayasu Taniguchi, Kazuhiko Besshi, Fumio Morita, Toshiki Kunihara, Fuminori Tanihara, Tatsuya Takemoto and Takeshige Otoi : Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes., Acta Veterinaria Hungarica, Vol.65, No.1, 115-123, 2017.
(Summary)
The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.
(Keyword)
Animals / DNA Damage / Hot Temperature / In Vitro Oocyte Maturation Techniques / Meiosis / Oocytes / Swine
Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
L Do, M Wittayarat, T Terazono, Y Sato, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, Y Kazuki, K Kazuki, M Oshimura and Takeshige Otoi : Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector., Reproduction in Domestic Animals = Zuchthygiene, Vol.51, No.6, 1039-1043, 2016.
N Kurniani Karja Wayan, M Fahrudin, MA Setiadi, LI Tumbelaka, R Sudarwati, YT Hastuti, BH Mulia, A Widianti, K Sultan, T Terazono, Z Namula, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, K Kikuchi, Y Sato and Takeshige Otoi : Characteristics and fertility of sumatran tiger spermatozoa cryopreserved with different sugars., Cryo Letters, Vol.37, No.4, 264-271, 2016.
(Summary)
Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.
M Nakai, J Ito, N Kashiwazaki, NT Men, Fuminori Tanihara, J Noguchi, H Kaneko, A Onishi and K Kikuchi : Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs, Theriogenology, Vol.85, No.4, 703-708, 2016.
(Summary)
To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.
Morita Yasuhiro, Taniguchi Masayasu, Fuminori Tanihara, Ito Aya, Namula Zhao, DO Thi Kim Lanh, Takagi Mitsuhiro, Tatsuya Takemoto and Takeshige Otoi : The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture, The Journal of Veterinary Medical Science, 2016.
(Summary)
The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0%, and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.
Ono Tetsushi, Isobe Tomohiro, Morita Yasuhiro, Do Lanh Thi Kim, Fuminori Tanihara, Taniguchi Masayasu, Takagi Mitsuhiro and Takeshige Otoi : Effects of parity and season on pregnancy rates after the transfer of embryos to repeat-breeder Japanese Black beef cattle., Archives Animal Breeding, Vol.59, 45-49, 2016.
(Summary)
<jats:p>Abstract. Repeat-breeder (RB) cows are a major source of economic waste due to their decreased fertility. Embryo transfer (ET) is an alternative tool to improve the fertility of RB cows. The aims of the present study were to evaluate the effects of recipient parity and the season on pregnancy rates following ET in RB Japanese Black beef cattle. Embryos were transferred nonsurgically to recipients, consisting of 155 heifers (< 2 years old) and 172 cows (< 8 years old), which were defined as RB cattle. Of the recipients that were presented for ET, 57 recipients received a fresh embryo and 270 recipients received a frozen embryo. There were no differences in the pregnancy rates between cattle that received fresh embryos or frozen embryos. The rates of recipients with pregnancy, abortion, stillbirth, and normal calving were similar between heifers and cows. In cows, the pregnancy rates were lower (P < 0.05) in summer (June to August) than in spring (March to May) and winter (December to February). In heifers, however, there were no differences in the pregnancy rates among the seasons. Our findings indicate that in RB Japanese Black beef cattle, the parity of the recipients does not have an effect on the pregnancy rates following the transfer of fresh and frozen embryos. However, heat stress may affect reproductive performance in RB Japanese Black cows. </jats:p>
Tetsushi Ono, Asako Takaoka, Yasuhiro Morita, Lanh Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : Effects of dibutyryl cyclic adenosine monophosphate and human chorionic gonadotropin on the formation of antral follicle-like structures by bovine cumulus-oocyte complexes., Acta Veterinaria Hungarica, Vol.63, No.4, 485-498, 2015.
(Summary)
This study evaluated the effect of dibutyryl cyclic adenosine monophos-phate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus- oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Müllerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.
L T. K. Do, Y Shibata, M Taniguchi, M Nii, T V. Nguyen, Fuminori Tanihara, M Takagi and Takeshige Otoi : Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.50, No.6, 1054-1058, 2015.
(Summary)
Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.
(Keyword)
Animals / Blastocyst / Culture Media / Embryonic Development / Female / Fertilization in Vitro / Melatonin / Oocytes / Swine
Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles., Animal Biotechnology, Vol.26, No.4, 273-275, 2015.
(Summary)
Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.
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.
(Summary)
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.
Tomohiro Isobe, Yoshihisa Ikebata, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer., Animal Science Journal, Vol.86, No.7, 661-665, 2014.
(Summary)
The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.
Manita Wittayarat, Akira Fujiwara, Kaywalee Chatdarong, Mongkol Techakumphu, Yoko Sato, Fuminori Tanihara, Yasuhiro Morita, Masayasu Taniguchi and Takeshige Otoi : Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors., Acta Veterinaria Hungarica, Vol.62, No.2, 233-242, 2014.
(Summary)
This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 μg/mL roscovitine or 0.05 μg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.
Tamás Somfai, Koji Yoshioka, Fuminori Tanihara, Hiroyuki Kaneko, Junko Noguchi, Naomi Kashiwazaki, Takashi Nagai and Kazuhiro Kikuchi : Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production., PLoS ONE, Vol.9, No.5, e97731, 2014.
(Summary)
We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.
(Keyword)
Animals / Blastocyst / Cell Culture Techniques / Cell Differentiation / Cell Survival / Cryopreservation / Embryo Transfer / Embryonic Development / Fertilization / Fertilization in Vitro / In Vitro Techniques / Oocytes / Swine / Vitrification
Hiroyuki Kaneko, Kazuhiro Kikuchi, Fuminori Tanihara, Junko Noguchi, Michiko Nakai, Junya Ito and Naomi Kashiwazaki : Normal reproductive development of pigs produced using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice., Theriogenology, Vol.82, No.2, 325-331, 2014.
(Summary)
Xenografting of immature testicular tissue combined with cryopreservation can preserve and use genetic information of prepubertal animals. For establishment of this new approach, it is essential to clarify whether offspring derived from sperm grown in host mice harboring cryopreserved xenografts show normal reproductive development. This study examined serum profiles of gonadal hormones during sexual maturation in pigs generated by intracytoplasmic sperm injection using sperm derived from cryopreserved xenografts (CryoXeno pigs; three males and three females). We also assessed the reproductive abilities of the male CryoXeno pigs by mating them with conventionally produced (conventional) pigs, and by examining the in vitro fertilizing ability of their sperm. For female CryoXeno pigs, reproductive ability was evaluated by artificial insemination with semen from a conventional boar. During the growth of male CryoXeno pigs, the serum concentrations of inhibin and testosterone showed similar changes (P > 0.17) to those in conventional pigs (n = 4). Histologic analyses of the testes revealed no differences (P > 0.2) in the growth and differentiation of seminiferous tubules between CryoXeno and conventional pigs. Three conventional sows delivered 13.0 ± 1.0 (mean ± standard error of the mean) live piglets after being mated with the three CryoXeno males. Sperm obtained from all CryoXeno pigs had the ability to penetrate oocytes, and these fertilized oocytes reached the blastocyst stage in vitro. During the growth of female CryoXeno pigs, the serum inhibin profile was similar (P > 0.17) to that observed in conventional pigs (n = 5). The first rise in serum progesterone concentration to more than 2 ng/mL was noted at 32.0 ± 2.3 weeks of age in the CryoXeno pigs and at 32.0 ± 3.3 weeks in the conventional pigs, suggesting that both pigs reached puberty at a similar age. After puberty, female CryoXeno pigs farrowed 8.3 ± 1.7 (mean ± standard error of the mean; n = 3) live piglets after artificial insemination with semen from a conventional boar. In conclusion, these findings demonstrate that both male and female CryoXeno pigs have normal reproductive abilities.
Zhao Namula, Risa Kodama, Fuminori Tanihara, Yasuhiro Morita, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi and Takeshige Otoi : Effects of skim-milk supplementation on the quality and penetrating ability of boar semen after long-term preservation at 15 °C., Acta Veterinaria Hungarica, Vol.62, No.1, 106-116, 2014.
(Summary)
This study investigated the effects of skim-milk supplementation on the quality and penetrating ability of boar semen preserved at 15 °C. When boar semen samples were preserved in Modified Modena extender supplemented with various concentrations (0, 7.5, 15, 30 and 50 mg/mL) of skim milk powder at 15 °C for 4 weeks, higher sperm motility and viability were observed in the case of 7.5 mg/mL skim-milk supplementation compared with the control group (0 mg/mL) during the preservation (P < 0.05). When in vitro matured oocytes were co-incubated with boar sperm that had been preserved in Modified Modena extender with three different concentrations (0, 7.5 or 15 mg/mL) of skim milk powder at 15 °C for two weeks, there were no apparent effects of skim-milk supplementation on the rates of fertilisation and development to blastocysts of oocytes after co-incubation. However, the monospermic fertilisation rate of sperm preserved with 15 mg/mL skim milk powder was higher (P < 0.05) than that of fresh non-preserved sperm, but did not differ among the preservation groups. The results indicate that the supplementation of Modified Modena extender with 7.5 mg/mL skim milk powder improves the motility and viability, but not the penetrating ability, of sperm after liquid preservation for at least two weeks.
Fuminori Tanihara, Michiko Nakai, Nguyen Thi Men, Noriko Kato, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi and Kazuhiro Kikuchi : Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs., Animal Science Journal, Vol.85, No.4, 395-404, 2014.
(Summary)
The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.
(Keyword)
Acrosome Reaction / Animals / Female / Fertilization / Fertilization in Vitro / Immunoglobulins / In Vitro Techniques / Male / Membrane Proteins / Oocytes / Sperm-Ovum Interactions / Spermatozoa / Swine / Zona Pellucida
Fuminori Tanihara, Yukine Kaedei, Zhao Namula, Vien Viet Luu, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi and Takeshige Otoi : Comparison of activation ability between feline and bovine oocytes., Acta Veterinaria Hungarica, Vol.61, No.4, 491-494, 2013.
(Summary)
Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.
Thi Nguyen Men, Kazuhiro Kikuchi, Michiko Nakai, Atsunori Fukuda, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Viet Nguyen Linh, Xuan Bui Nguyen, Takashi Nagai and Atsushi Tajima : Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection., Theriogenology, Vol.80, No.9, 1033-1044, 2013.
(Summary)
Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI.
(Keyword)
Animals / DNA Breaks, Double-Stranded / Embryonic Development / Fertilization / Freeze Drying / Male / Sperm Injections, Intracytoplasmic / Spermatozoa / Swine / Trehalose
István Egerszegi, Tamás Somfai, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Takashi Nagai, József Rátky and Kazuhiro Kikuchi : Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model., Cryobiology, Vol.67, No.3, 287-292, 2013.
(Summary)
Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.
(Keyword)
Animals / Cell Survival / Cryopreservation / Cytoskeleton / Embryonic Development / Female / Fertilization in Vitro / Male / Oocytes / Swine / Vitrification
Hiroyuki Kaneko, Michiko Nakai, Fuminori Tanihara, Junko Noguchi and Kazuhiro Kikuchi : Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes., Theriogenology, Vol.80, No.8, 887-892, 2013.
(Summary)
Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.
Nguyen Linh Viet, Kazuhiro Kikuchi, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Quang Thanh Dang-Nguyen, Thi Nguyen Men, Nguyen Hanh Van, Tamas Somfai, Xuan Bui Nguyen, Takashi Nagai and Noboru Manabe : Fertilization ability of porcine oocytes reconstructed from ooplasmic fragments produced and characterized after serial centrifugations., The Journal of Reproduction and Development, Vol.59, No.6, 549-556, 2013.
(Summary)
Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.
(Keyword)
Abattoirs / Animals / Animals, Inbred Strains / Cell-Free System / Centrifugation, Density Gradient / Crosses, Genetic / Cryopreservation / Cytoplasmic Structures / Electrochemical Techniques / Female / Fertilization in Vitro / In Vitro Oocyte Maturation Techniques / Japan / Male / Membrane Fusion / Mitochondria / Oocytes / Sperm-Ovum Interactions / Spermatozoa / Sus scrofa / Up-Regulation
Hiroyuki Kaneko, Kazuhiro Kikuchi, Michiko Nakai, Tamas Somfai, Junko Noguchi, Fuminori Tanihara, Junya Ito and Naomi Kashiwazaki : Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice., PLoS ONE, Vol.8, No.7, e70989, 2013.
(Summary)
Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.
Fuminori Tanihara, Michiko Nakai, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi and Kazuhiro Kikuchi : Evaluation of zona pellucida function for sperm penetration during in vitro fertilization in pigs., The Journal of Reproduction and Development, Vol.59, No.4, 385-392, 2013.
(Summary)
In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP- oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP- oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP- oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP- oocytes at 1-10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP- oocytes. Finally, we performed IVF using ZP- oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.
(Keyword)
Animals / Female / Fertilization in Vitro / Male / Oocytes / Sperm-Ovum Interactions / Swine / Zona Pellucida
Tamás Somfai, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Naomi Kashiwazaki, István Egerszegi, Takashi Nagai and Kazuhiro Kikuchi : Comparison of ethylene glycol and propylene glycol for the vitrification of immature porcine oocytes., The Journal of Reproduction and Development, Vol.59, No.4, 378-384, 2013.
(Summary)
Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.
(Keyword)
Animals / Cryopreservation / Cryoprotective Agents / Embryonic Development / Ethylene Glycol / Female / Fertilization in Vitro / Male / Oocytes / Propylene Glycol / Swine / Vitrification
Vien Viet Luu, Keisuke Hanatate, Fuminori Tanihara, Yoko Sato, Lanh Thi Kim Do, Masayasu Taniguchi and Takeshige Otoi : The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4°C., Reproductive Biology, Vol.13, No.2, 122-126, 2013.
(Summary)
Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4°C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40ng/ml) of relaxin for 24h. They were then fertilized in vitro for 12h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10ng/ml relaxin (16.0%) was higher (p<0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.
(Keyword)
analysis of variance / Animals / Cats / Cryopreservation / Culture Media / Dose-Response Relationship, Drug / Embryo Culture Techniques / Embryonic Development / Female / Oocytes / ovary / Relaxin / Reproductive Techniques, Assisted
Zhao Namula, Yoko Sato, Risa Kodama, Kouta Morinaga, Viet Vien Luu, Masayasu Taniguchi, Michiko Nakai, Fuminori Tanihara, Kazuhiro Kikuchi, Takashi Nagai and Takeshige Otoi : Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks., Animal Science Journal, Vol.84, No.8, 600-606, 2013.
(Summary)
This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.
(Keyword)
Animals / Cold Temperature / Fertilization in Vitro / Male / Semen / Semen Analysis / Semen Preservation / Sperm Motility / Swine / Time Factors
Hiroyuki Kaneko, Kazuhiro Kikuchi, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Michiko Noguchi, Junya Ito and Naomi Kashiwazaki : Normal reproductive development of offspring derived by intracytoplasmic injection of porcine sperm grown in host mice., Theriogenology, Vol.78, No.4, 898-906, 2012.
(Summary)
For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities.
Y Kaedei, M Naito, H Naoi, Y Sato, M Taniguchi, Fuminori Tanihara, K Kikuchi, T Nagai and Takeshige Otoi : Effects of (-)-epigallocatechin gallate on the motility and penetrability of frozen-thawed boar spermatozoa incubated in the fertilization medium., Reproduction in Domestic Animals = Zuchthygiene, Vol.47, No.6, 880-886, 2012.
(Summary)
Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen-thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 m EGCG for 1, 3 and 5 h, supplementation with 50 and 100 m EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen-thawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 m EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen-thawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 m EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 m EGCG, but the effects vary with individual boars.
(Keyword)
Animals / catechin / Cryopreservation / Dose-Response Relationship, Drug / Female / Fertilization in Vitro / Male / Semen Preservation / Sperm Motility / Sperm-Ovum Interactions / Spermatozoa / Swine
Masayasu Taniguchi, Rie Arikawa, Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Yoko Sato and Takeshige Otoi : Effects of cryoprotectant agents and equilibration methods on developmental competence of porcine oocytes., Cryo Letters, Vol.32, No.5, 410-414, 2011.
(Summary)
Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine oocytes was examined. In vitro-matured oocytes were exposed to 40 percent ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured for 8 days. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40 percent EG by 3 multi-step methods, after which their developmental competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.
T Terazono, Y Kaedei, Fuminori Tanihara, Z Namula, Vl Viet, M Takagi, M Inoue, Y Sato, M Taniguchi and Takeshige Otoi : Follicle formation in the canine ovary after autografting to a peripheral site., Reproduction in Domestic Animals = Zuchthygiene, Vol.47, No.2, e16-21, 2011.
(Summary)
This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.
T Terazono, M Inoue, Y Kaedei, Fuminori Tanihara, Z Namula, L V Viet, Y Taura, M Takagi, T Takuma and Takeshige Otoi : Assessment of canine ovaries autografted to various body sites., Theriogenology, Vol.77, No.1, 131-138, 2011.
(Summary)
The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary.
Masao Murakami, Juan Ya Dong, Tatsuyuki Suzuki, Masayasu Taniguchi, Yukine Kaedei, Yoko Sato, Fuminori Tanihara and Takeshige Otoi : Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media., Cryobiology, Vol.63, No.3, 170-174, 2011.
(Summary)
The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P<0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.
V E Abakushina, Y Morita, Y Kaedei, Fuminori Tanihara, Z Namula, L V Viet and Takeshige Otoi : Formation of an antral follicle-like structure of bovine cumulus-oocyte complexes embedded individually or in groups in collagen gels., Reproduction in Domestic Animals = Zuchthygiene, Vol.46, No.3, 423-427, 2010.
(Summary)
Culture techniques of antral follicle-like structure (AFLS) derived from cumulus-oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4-5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day=0), irrespective of the culture methods. The mean diameters of AFLS during Days 4-14 using the individual culture method were significantly higher (p<0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p<0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.
Yavari Morteza, Naoi Hideaki, Kaedei Yukine, Fuminori Tanihara, Namula Zhao, Viet Luu Vien and Takeshige Otoi : Effects of epigallocatechin- 3-gallate on the developmental competence of parthenogenetic embryos in the pig, Italian Journal of Animal Science, Vol.9, No.4, 386-389, 2010.
(Summary)
This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 µM). Supplementation of 1 and 5 µM EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 µM EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.
Kaedei Yukine, Fujiwara Akira, Ito Aya, Fuminori Tanihara, Morita Yasuhiro, Hanatate Keisuke, Viet Luu Vien, Namula Zhao and Takeshige Otoi : Effect of Roscovitine Pretreatment on the Meiotic Maturation of Bovine Oocytes and their Subsequent Development after Somatic Cell Nuclear Transfer, Journal of Animal and Veterinary Advances, Vol.9, No.22, 2848-2853, 2010.
(Summary)
Roscovitine, a specific inhibitor of M-phase promoting factor kinase activity was used to inhibit the completion of meiotic maturation of bovine oocytes. The objectives of this study were to evaluate the nuclear maturation of bovine oocytes pre-cultured with various concentrations (0, 50, 100 and 200 μM) of roscovitine before in vitro Maturation (IVM) and to examine the development of Somatic Cell Nuclear Transfer (SCNT) embryos derived from the oocytes pre-cultured with roscovitine. Before IVM, 72% of oocytes that were cultured without roscovitine (control) had reached the Metaphase II (MII) stage whereas culture with roscovitine decreased the rates of oocytes reaching MII (11-27%). After IVM, the maturation rate of oocytes pre-cultured with 200 μM roscovitine was significantly higher than that of control oocytes (79 vs. 58%). Moreover, significantly more oocytes extruded the first polar body in the 50 μM roscovitine group than in the control group (64 vs. 51%). The rate of blastocyst formation of reconstructed embryos derived from oocytes pre-cultured with 50 μM roscovitine was significantly higher than that from the control oocytes (14 vs. 6%). In this study, the addition of roscovitine to culture medium delays the completion of meiotic maturation of bovine oocytes and the cytoplasm derived from oocytes pre-cultured under meiotic inhibition can support the development of SCNT embryos.
Y Kaedei, A Fujiwara, Fuminori Tanihara, Z Namula, VL Viet and Takeshige Otoi : In vitro development of cat interspecies nuclear transfer using pig's and cow's cytoplasm, Bulletin of the Veterinary Institute in Puławy, Vol.54, No.3, 405-408, 2010.
A Fujii, Y Kaedei, Fuminori Tanihara, A Ito, K Hanatate, K Kikuchi, T Nagai and Takeshige Otoi : In vitro maturation and development of porcine oocytes cultured in a straw or dish using a portable incubator with a CO2 chamber., Reproduction in Domestic Animals = Zuchthygiene, Vol.45, No.4, 619-624, 2009.
(Summary)
We investigated the effects of a portable incubator with a CO(2) chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus-oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO(2) incubator, in the CO(2) chamber in an incubator, or in the CO(2) chamber in a portable incubator. The matured oocytes were fertilized with frozen-thawed spermatozoa and then cultured in a dish in the standard CO(2) incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO(2) incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO(2) incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO(2) chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.
Agung Budiyanto, Barati Farid, Kaedei Yukine, Fuminori Tanihara and Takeshige Otoi : Meiotic Competence and DNA Damage of Porcine Oocytes from Ovaries Exposed to an Elevated Temperature, Journal of Animal and Veterinary Advances, Vol.7, No.10, 1179-1183, 2008.
(Summary)
The present study was conducted to investigate the effects of the exposure length of ovaries to an elevated temperature (41°C) on the meiotic competence and DNA damage of oocytes. Ovaries were stored in physiological saline at 41°C for 0 h (control), 0.5, 1.0 and 1.5 h. After exposure of ovaries to the elevated temperature, oocytes were collected and then cultured for 44 h. The length of exposure of ovaries to 41°C had no effect on the proportions of total oocytes with DNA-fragmented nuclei before maturation culture, but it did influence the proportions at the end of maturation culture. The proportion of oocytes reaching metaphase II (MII) significandy decreased with increasing exposure time. In addition, significantly more oocytes from ovaries exposed to 41°C for 1.5 h had DNA-fragmented nuclei compared with control oocytes. These results indicate that the meiotic competence and DNA damage of porcine oocytes are dependent on the duration of exposure of ovaries to the elevated temperature. Moreover, the occurrence of DNA damage in oocytes becomes more apparent after maturation culture than before the culture.
M. Yavari, A. Fujii, R. Shimizu, A. Ito, Y. Kaedei, Y. Morita, Fuminori Tanihara and Takeshige Otoi : Meiotic competence of porcine oocytes after percoll sedimentation treatment for oocyte selection., Journal of Animal Veterinary advances, Vol.6, No.11, 1333-1336, 2007.
Review, Commentary:
1.
Fuminori Tanihara, Maki Hirata and Takeshige Otoi : ゲノム編集技術による遺伝子改変家畜の作出, Journal of Mammalian Ova Research, Vol.39, 17-26, Jan. 2022.
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.
Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : Effects of CRISPR/Cas9-mediated gene targeting of porcine endogenous retrovirus on the developmental competence of porcine embryos, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
4.
Nguyen Thi Nhien, Fuminori Tanihara, Maki Hirata, Hirano Takayuki, Le Anh Quynh and Takeshige Otoi : Efficiency of gene editing by electroporation of Cas9 protein (GEEP) to generate GGTA1-modified pigs, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
5.
Le Anh Quynh, Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Hirano Takayuki and Takeshige Otoi : Effect of Cas9 protein levels on genomic mutations using the gene editing by electroporation of Cas9 protein (GEEP) system in putative zygotes, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
6.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : Assessment of PDX-1-deficient pigs generated using the CRISPR/Cas9 system introduced into porcine zygotes via electroporation, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
7.
Akio Kuroda, Misuzu Yamada, Yukari Tominaga, Reiko Suzuki, Motoyuki Tamaki, Yuko Akehi, Yuichi Takashi, Daisuke Otsuka, Eisuke Shimokita, Fuminori Tanihara, Kiyoe Kurahashi, Sumiko Yoshida, Itsuro Endo, Ken-ichi Aihara, Masahiro Abe, Kevin Ferreri and Munehide Matsuhisa : Detection of pancreatic beta cell DNA in the circulation using the amplification refractory mustation system PCR, American Diabetes Association 78th Scientific Sessions, Orlando, Jun. 2018.
8.
Katsutoshi Nishio, Fuminori Tanihara, TV Nguyen, Toshiki Kunihara and Takeshige Otoi : Effects of voltage strength on development and quality of electroporated porcine embryos., Transgenic Research, Vol.26, No.1, 29, Utah, USA, Oct. 2017.
9.
Fuminori Tanihara, LTK Do, TV Nguyen, Toshiki Kunihara, Katsutoshi Nishio, Tatsuya Takemoto and Takeshige Otoi : Generation of TP53-modified pigs by GEEP method: CRISPR/Cas9-mediated gene modification introduced into porcine zygotes by electroporation., Transgenic Research, Vol.26, No.1, 38, Utah, USA, Oct. 2017.
10.
Fuminori Tanihara and Takeshige Otoi : A simple-step generation of genetically modified pigs by genome editing by electroporation of Cas9 protein (GEEP) method., Forth World Congress of Reproductive Bioligy (WCRB2017), Okinawa, Sep. 2017.
(Summary)
Pigs are considered as one of the best animals for generating models of human diseases and for providing organs for xenotransplantation. Currently, genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer (SCNT) technique after the generation of engineered donor somatic cells. However, this approach requires complex micromanipulation techniques and a long time for preparing enough number of reconstructed embryos to be transferred to the surrogate pig. Recently, we have established a simple method for CRISPR /Cas9 gene editing that involves the introduction of Cas9 protein and single-guide RNA into in vitro-fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. While the genotype of genetically modified pig generated using the SCNT is homogeneous, the introduction of CRISPR/Cas9 system into embryos may induce mosaic genotype. However, GEEP method will be a powerful tool to achieve gene modification in the pig. We are now applying this method to generate various kinds of gene-modified pigs as an intractable disease model. We introduce recent studies and trials for generating genetically modified pigs by GEEP method.
11.
R Kuriwaki, Y Sato, S Hagino, M Shimazaki, R Sambuu, LTK Do, Fuminori Tanihara, M Takagi, M Taniguchi and Takeshige Otoi : Evaluation of the Leydig cell function on crossbreeding yak showing infertility., Forth World Congress of Reproductive Bioligy (WCRB2017), Okinawa, Sep. 2017.
Q Lin, Quynh Anh Le, Manita Wittayarat, Zhao Namula, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Kim Lanh Thi Do and Takeshige Otoi : Triple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods., 第7回ゲノム編集学会, Jun. 2022.
3.
Lin Qingyi, Chommanart Thongkittidilok, Maki Hirata, QUYNH ANH LE, K. Takebayashi, Fuminori Tanihara and Takeshige Otoi : Lipofection-mediated introduction of CRISPR/Cas9 system into porcine zygotes., 第114回日本繁殖生物学会, Sep. 2021.
4.
TAKEBAYASHI Kohki, Chommanart Thongkittidilok, 林 青怡, LE Anh Quynh, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : ノックアウトブタ由来精子を用いた体外受精胚におけるゲノム編集効率の検討, 第114回日本繁殖生物学会, Sep. 2021.
5.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Quynh Anh Le, Takayuki Hirano and Takeshige Otoi : エレクトロポレーションを用いたCRISPR/Cas9システムのブタ体外受精卵への導入によるCD163遺伝子改変ブタの作製, 第162回日本獣医学会学術集会, Sep. 2019.
ブタは生理学的および解剖学的にヒトに近い優れたモデル動物である．これまで遺伝子改変ブタの作製は，遺伝子改変を行った体細胞を用いた核移植によるクローンブタの作製によりなされてきた(体細胞クローン法)．さらに近年Crispr/Cas9システムをはじめとするゲノム編集技術が体細胞の遺伝子改変に活用されている．体細胞クローン法ではbiallelic変異個体を一代で得られるという利点があるが，遺伝子改変細胞株の樹立からクローン胚作製と高度な手技および時間が必要であり，さらに正常産仔の作製効率も未だ低く，実施できる研究者・研究機関は限られている．この問題を解決するため，発表者らはブタ体外受精卵に対しエレクトロポレーションにより CRISPR/Cas9 システムを導入し，簡便かつ高効率に遺伝子改変を行うGEEP法(Genome editing by electroporation of Cas9 protein)を確立した．この手法によりすでに複数のゲノム編集ブタ作製に成功しているが，得られたゲノム編集ブタの解析から，biallelic変異個体の作製に成功しているもののwt配列の残るモザイク個体も存在することがわかり，GEEP法においてモザイク個体率を下げることが課題となっている．今回，ブタ体外受精卵および産仔について，ゲノム編集ブタ作製に使用するガイドRNAとエレクトロポレーションによる導入条件，およびモザイク率の関連を複数の標的遺伝子で解析した．その結果，適切なガイドRNAおよびエレクトロポレーション条件の選択により，biallelic変異個体を一代で得られる可能性が示唆された．
近年，再生医療技術およびゲノム編集技術の進展に伴い，ブタの組織・臓器を用いた異種移植が現実味を帯びてきた．一方，ブタゲノム中にはブタ内在性レトロウイルス(PERV)が多数内在されており，移植後におけるヒトへの感染が懸念されている．ゲノム編集技術により，ブタゲノム中に存在するPERV遺伝子をノックアウトすることにより，PERV感染の制御が可能と考えられるが，内在性レトロウイルスの初期発生との関連性，特に，初期胚発育能に及ぼす影響は未知数である．我々は，これまでにエレクトロポレーションによりCRISPR/Cas9システムをブタの1細胞期体外受精卵に導入することで，ゲノム編集を高効率に行い，かつ胚の生存性を維持できるGEEP法(genome editing by electroporation of Cas9 protein)を確立した．本研究ではブタの体外受精卵において，細胞内でPERV 自身を複製する際に重要なpol 遺伝子を標的にGEEP法によりノックアウトし，ゲノム編集効率と胚発育の関連性を調査した．pol遺伝子を標的とするguideRNA(gRNA)を5種類作製し，それぞれのgRNAをCas9タンパク質とともにブタの体外受精卵に導入した．その結果，一部のgRNA導入群において胚盤胞形成率が非常に低い値を示した．これらの群において4-8細胞期のゲノム編集効率を検討したところ，編集効率は他の群よりも高い値を示し，ゲノム編集効率と胚盤胞形成率に負の関連性が認められた．このことから，PERV遺伝子は胚発生と関連していることが示唆された．
ブタは生理学的，解剖学的性質や生物学的特性がヒトに近く，実験動物として注目されている．ヒトの病態モデルとなるようなブタを遺伝子改変技術によって作製することができれば，難病疾患の研究や，創薬研究，また，手術トレーニングに活用することができ，医学研究の大幅な発展が期待される．従来遺伝子改変ブタの作製は，遺伝子改変を行った体細胞を用いた核移植によるクローンブタの作製によりなされてきた(体細胞クローン法)．ゲノム編集技術の登場により，受精胚細胞質へのマイクロインジェクション法による遺伝子改変ブタの作製も可能となったが，体細胞クローン法とマイクロインジェクション法は共に高度な技術が必要であることから，実施できる研究者・研究機関は限られている． 私たちは，エレクトロポレーションにより CRISPR/Cas9 システムをブタ体外受精胚に導入することで，簡便かつ高効率に遺伝子改変を行うGEEP法(genome editing by electroporation of Cas9 protein)を確立し，本法を用いて遺伝子改変ブタの作製に成功した．GEEP法は高度な技術を必要とせず，簡単な操作で受精胚のゲノム編集が可能である．本セミナーでは，エレクトロポレーションを用いたゲノム編集ブタの作製について詳しく紹介したい．
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : CRISPR/Cas9システムによるブタ体外受精卵のINS遺伝子への点変異導入, 第7回日本先進医工学ブタ研究会, Oct. 2019.
3.
Takeshige Otoi, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Fuminori Tanihara : GEEP法を用いた遺伝子改変ブタの作製と遺伝子改変効率, 第7回日本先進医工学ブタ研究会, Oct. 2019.
4.
Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : ブタ体外受精卵におけるCRISPR/Cas9システムを用いた複数遺伝子の同時改変, 第7回日本先進医工学ブタ研究会, Oct. 2019.
5.
Le Anh Quynh, Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Takayuki Hirano and Takeshige Otoi : Concentration of CRISPR/Cas9 components effects on genetic mosaicism of cytoplasmic microinjected porcine embryos, 第6回日本先進医工学ブタ研究会, Oct. 2018.
(Summary)
Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs, but mosaicism is a serious problem for genetically modified pigs because of the complicated analysis of phenotypes. In the present study, we examined the suitable timing and concentration of CRISPR/Cas9 for introduction into oocytes/zygotes by CI to reduce mosaicism in the resulting blastocysts. In the first experiment, we introduced 20 ng/μl of Cas9 protein and gRNA, targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes and zygotes before and after IVF twice. CI treatment had no detrimental effects on the blastocyst formation rates, but the number of mutant blastocysts from zygotes injected after IVF tended to increase compared to that from oocytes injected before IVF (p < 0.1). In the second experiment, we injected 20 ng/μl or 100 ng/μl of Cas9 protein and gRNA into zygotes after IVF. Although no blastocysts that were injected with 20 ng/μl of Cas9 protein and gRNA carried a biallelic mutation, 50% of blastocysts that were injected with 100 ng/μl of Cas9 protein and gRNA carried a biallelic mutation. In conclusion, to achieve efficient gene editing in porcine zygotes by CI of Cas9 protein with gRNA, performing CI after IVF is better than performing CI before IVF. Furthermore, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI and the concentration of CRISPR/Cas9 components is needed.
6.
Nguyen Thi Nhien, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Le Anh Quynh, 新居 雅宏 and Takeshige Otoi : Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid, 第6回日本先進医工学ブタ研究会, Oct. 2018.
(Summary)
The current study was conducted to investigate the effects of 100% fetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 h. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25°C for 24 h in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/mL bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 h was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.
7.
Maki Hirata, Fuminori Tanihara, Takayuki Hirano, Nhien Thi Nguyen, Quynh Anh Le, 新居 雅宏 and Takeshige Otoi : ブタにおける受精前後でのゲノム編集が胚盤胞の変異導入効率に及ぼす影響, 第6回日本先進医工学ブタ研究会, Oct. 2018.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Takayuki Hirano, Tatsuya Takemoto, 中井 美智子, 淵本 大一郎 and Takeshige Otoi : ゲノム編集によるTP53遺伝子改変ブタの作製と表現型の解析, 第6回日本先進医工学ブタ研究会, Oct. 2018.
9.
Fuminori Tanihara, Tatsuya Takemoto, Maki Hirata, N Nguyen Thi, Toshiki Kunihara, R Nishinakamura and Takeshige Otoi : Modification of SALL1 gene via CRISPR/Cas9-mediated gene editing introduced into porcine zygotes by electroporation, KEY Forum: The 3rd International symposium on Stem Cell Traits and Developmental Systems, Jan. 2018.
10.
Fuminori Tanihara and Takeshige Otoi : ゲノム編集技術を用いたブタでの応用例, 第3回日本生物工学会西日本支部講演会「ゲノム編集—多様な生物種への応用研究」, Dec. 2016.
11.
Fuminori Tanihara, Tatsuya Takemoto, Michiko Nakai, Eri Kitagawa, Do Thi Kim Lanh, Akira Onishi, Shunichi Suzuki, Syoichiro Senbon, Daiichiro Fuchimoto and Takeshige Otoi : 新規ゲノム編集技術を用いたPDX-1遺伝子改変ブタの作製, 第4回 日本先進医工学ブタ研究会 要旨集, 21, Oct. 2016.