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S Haraguchi,
T Tokunaga,
T Furusawa,
K Ohkoshi,
M Nakai,
M Ikeda, K Kikuchi,
T Q Dang-Nguyen,
T Somfai,
S Akagi,
M Kaneda,
Y Hirao,
S Watanabe,
M Geshi,
T Nagai
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ABSTRACT: Despite meticulous attempts for more than two decades, establishment of authentic porcine embryonic stem cell (ESC) from pig has never been successful. Although putative porcine ESC-like cells have been reported, such cell lines easily lose the ability of self-renewal, becoming extinct or differentiating after only a limited number of passages in culture. Porcine ESC-like cells exhibiting the property of self-renewal rather than pluripotency are considered a valuable resource in applications such as drug screening and toxicology testing in humans and livestock, and in veterinary medicine. In the present study, we evaluated the effect of glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 and Erk signalling inhibitor PD184352 for use in establishing ESC-like cell lines derived from the inner cell mass (ICM) of porcine blastocysts produced in vitro. These ICM-derived cell lines were initially cultured and passaged in conventional human ES medium. They displayed so-called ESC-like morphology; for example, the isolated colonies began to grow as a monolayer with coarse cell-cell boundaries, in which the cells exhibited polygonal boundaries, high nuclear/cytoplasmic ratios, abundant lipid-like inclusions, alkaline phosphatase activity, and expression of markers of undifferentiated cells such as OCT4 and NANOG. After transfer to culture in ES medium containing the inhibitors, the morphology of the colony was dramatically changed, displaying a closely packed and smooth-edged colony with tight cell-cell boundaries. Remarkably, growth of the established cell lines is leukemia inhibitory factor (LIF)-dependent. The inclusion of inhibitors supports self-renewal, thus enabling continuous culture for over 100 passages while maintaining an undifferentiated state. High-passage-number cells continued to express undifferentiated marker genes and showed alkaline phosphatase activity and telomerase activity with an X chromosome status of X(a)X(i). We further investigated the potential for differentiation of the established cell lines. The cells could easily form embryoid body-like spheres in suspension culture. When either the spheres or ESC-like cells were inoculated under the kidney or testis capsules of nude mice, classical teratoma formation was not observed after 2 to 3 months. However, histological analyses revealed apparent invasive proliferation derived from porcine cells. Although further analyses are required to characterise the property of the porcine ESC-like cells, we have recently succeeded in establishment of green fluorescent protein (GFP)-expressing stable cells lines, which will be useful for further investigation.
Reproduction Fertility and Development 12/2012; 25(1):297. · 2.11 Impact Factor
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ABSTRACT: We compared the feasibility of ethylene glycol (EG) and propylene glycol (PG) for the vitrification of immature porcine cumulus-oocyte complexes (COC). Porcine COC collected from 3- to 6-mm follicles of slaughterhouse-derived ovaries were subjected to solid-surface vitrification (Somfai et al. 2010 Theriogenology 73, 147-156) either in 35% (v/v) EG or 35% (v/v) PG or in the mixture of 17.5% (v/v) EG and 17.5% (v/v) PG. After warming, the COC were subjected to in vitro maturation, IVF, and embryo culture according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033-1041). Oocyte survival and maturation rates were assessed after in vitro maturation by evaluating membrane integrity and the extrusion of the first polar body. All live oocytes were subjected to IVF and in vitro culture. Cleavage and blastocyst rates were calculated from the total number of oocytes subjected to IVF on Day 2 (Day 0=IVF) and Day 7, respectively. Total-cell (blastomeres) numbers in blastocysts were recorded on Day 7 after staining with Hoechst 33342. In Experiment 1, competence parameters of oocytes vitrified either in EG-based (EG group; n=310) or a PG-based (PG group; n=265) vitrification media were compared with those in the nonvitrified control (n=160). The experiment was replicated 4 times. In Experiment 2, the competence parameters of oocytes vitrified with the combination of 17.5% EG and 17.5% PG (EG+PG group; n=397) were compared with those in nonvitrified control (n=245) and toxicity control (TC, exposed to cryoprotectants without cooling; n=245) groups. Five replications were performed. Results were analyzed by ANOVA. Differences with P<0.05 were considered significant. In Experiment 1, the mean survival rate of vitrified oocytes was significantly higher (P<0.05) in 35% PG compared with that in 35% EG (73.3 and 25.9%, respectively). Maturation rates of surviving oocytes did not differ among vitrified (PG and EG) and nonvitrified control groups (71.1, 62.4, and 64.0%, respectively). After IVF of surviving oocytes, blastocyst formation rate in the group vitrified in EG was higher (P<0.05) compared with that vitrified in PG but was lower (P<0.05) compared with control (10.8, 2.0, and 25.0%, respectively). Mean cell numbers in blastocysts did not differ among EG, PG, and control groups (50.5, 47.7, and 48.7, respectively). In Experiment 2, survival of immature oocytes in the EG+PG group was 42.6%. After IVF, 10.7% of oocytes developed to the blastocyst stage in the EG+PG group, which was lower (P<0.05) than those of the control (18.1%) and TC (23.3%) groups. Blastocyst rates in the control and TC groups were not statistically different. Mean cell numbers in blastocysts did not differ significantly among the EG+PG, control, and TC groups (61.6, 59.3, and 53.3, respectively). In conclusion, 35% PG provided a higher oocyte survival rate after vitrification compared with 35% EG. However, presumably due to toxic effects, 35% PG greatly reduced the development competence of oocytes. The combination of 17.5% EG and 17.5% PG yielded higher survival rates than did 35% EG, without any toxic effect on oocytes.
Reproduction Fertility and Development 12/2012; 25(1):187. · 2.11 Impact Factor
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ABSTRACT: Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8±1.5kb), C (24.4±0.5kb), P7 (18.7±0.6kb), and P10 (17.2±0.1kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2±0.3, 17.8±0.7, 18.5±0.3, and 18.4±0.4kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3±1.5, 23.5±2.6, 20.2±1.0, and 20.9±1.0kb, respectively). However, the telomere of the blastocyst was significantly longer (P<0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2±0.4, 4.0±0.5, 5.1±0.4, and 4.9±0.4, respectively) were significantly lower (P<0.01) than those of cloned blastocysts in the same groups (8.2±1.1, 8.6±0.6, 12.5±2.9, and 8.3±1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0±5.2, 17.3±2.9, 13.5±2.9, and 13.1±3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3±2.9, 32.6±3.6, 30.4±3.1, and 27.4±2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.
Reproduction Fertility and Development 12/2012; 25(1):166. · 2.11 Impact Factor
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ABSTRACT: We develop a highly efficient quasi-phase-matched adhered ridge waveguide (QPM-ARW) in LiNbO $_3$ as a nonlinear material, and demonstrate tunable wavelength conversion without spectral inversion (SI) and parametric tunable dispersion compensation for a single-mode fiber (SMF) link. The QPM-ARW module with a second harmonic generation efficiency of 700 %/W achieves tunable wavelength conversion with a wavelength-tuning range of at least 25 nm through cascaded sum- and difference-frequency generation (SFG-DFG) process in which the signal and pumps are located symmetrically around the phase matching wavelength. The power penalty of the wavelength conversion is less than 0.6 dB for 43-Gb/s nonreturn-to-zero on–off-keying (NRZ-OOK) signals. We then apply the tunable wavelength conversion without SI to the parametric tunable dispersion compensation scheme, and achieve successful optical tunable dispersion compensation in 43-Gb/s NRZ-OOK transmissions over 53.2-km SMF.
IEEE Journal of Selected Topics in Quantum Electronics 05/2012; · 3.78 Impact Factor
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T Somfai, K Kikuchi,
M Kaneda,
S Akagi,
S Watanabe,
E Mizutani,
S Haraguchi,
T Q Dang-Nguyen,
Y Inaba,
M Geshi,
T Nagai
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ABSTRACT: We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.
Anantomia Histologia Embryologia 05/2011; 40(5):335-44. · 0.90 Impact Factor
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T Q Dang-Nguyen,
M Kaneda,
T Somfai,
S Haraguchi,
K Matsukawa,
S Akagi, K Kikuchi,
M Nakai,
B X Nguyen,
A Tajima,
Y Kanai,
T Nagai
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ABSTRACT: The objective was to investigate development of single blastomeres derived from IVP two-cell porcine embryos. There was no difference (P > 0.05) in blastocyst rates among intact two-cell embryos (IN), zona-free two-cell embryos (ZF), and single blastomere (SB) groups (50.0 ± 9.7, 57.4 ± 5.7, and 45.1 ± 7.2%, respectively; mean ± SEM). However, blastocyst yield for the SB group (90.2 ± 14.4%, based on the original number of two-cell embryos before blastomere separation) was higher (P < 0.05) than those of IN and ZF groups. Although the number of inner cell mass (ICM) and trophectoderm (TE) cells in SB blastocysts (6.2 ± 0.8 and 15.5 ± 1.1, respectively) was lower (P < 0.05) than those in IN (12.4 ± 1.3 and 26.0 ± 3.8) and ZF blastocysts (10.7 ± 1.6 and 26.4 ± 3.4), ICM:TE ratios did not differ significantly among groups. Expressions of transcripts associated with cellular organization (TUBA1 and TUBB) were reduced (P < 0.05) in SB versus IN blastocysts. However, there was no significant difference among groups for expression of transcripts associated with responses to stress (HSPE1, HSPD1, and HSPCA) or glucose catabolism (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts. The percentage of the sister blastomere pairs in which both cells developed to blastocysts (36.6 ± 5.3%) or both degenerated (46.3 ± 10.3%) were higher (P < 0.05) than that of the pairs in which one developed to blastocyst while the other degenerated (17.1 ± 7.8%). When both pairs developed to blastocysts, one blastocyst had more (P < 0.05) ICM and TE cells (8.2 ± 1.2 and 20.2 ± 2.1, respectively) than the other (5.2 ± 0.9 and 13.5 ± 1.1), although ICM:TE cell ratios were not significantly different. In conclusion, blastomere separation at the two-cell stage significantly increased blastocyst yield from IVP porcine embryos. This might be a useful approach for conservation of rare pig breeds, in which low numbers of embryos limited the success of embryo transfer.
Theriogenology 03/2011; 76(1):88-96. · 1.96 Impact Factor
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Dang-Nguyen Quang Thanh,
M Kaneda,
T Somfai,
K Matsukawa,
S Akagi, K Kikuchi,
Bui Xuan Nguyen,
M Nagai,
A Tajima,
Y Kanai,
Takashi Nagai
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ABSTRACT: Abstract
The objective was to investigate development of single blastomeres derived from IVP two-cell porcine embryos. There was no difference (P > 0.05) in blastocyst rates among intact two-cell embryos (IN), zona-free two-cell embryos (ZF), and single blastomere (SB) groups (50.0 ± 9.7, 57.4 ± 5.7, and 45.1 ± 7.2%, respectively; mean ± SEM). However, blastocyst yield for the SB group (90.2 ± 14.4%, based on the original number of two-cell embryos before blastomere separation) was higher (P < 0.05) than those of IN and ZF groups. Although the number of inner cell mass (ICM) and trophectoderm (TE) cells in SB blastocysts (6.2 ± 0.8 and 15.5 ± 1.1, respectively) was lower (P < 0.05) than those in IN (12.4 ± 1.3 and 26.0 ± 3.8) and ZF blastocysts (10.7 ± 1.6 and 26.4 ± 3.4), ICM:TE ratios did not differ significantly among groups. Expressions of transcripts associated with cellular organization (TUBA1 and TUBB) were reduced (P < 0.05) in SB versus IN blastocysts. However, there was no significant difference among groups for expression of transcripts associated with responses to stress (HSPE1, HSPD1, and HSPCA) or glucose catabolism (ENO1, COX6C, COX7B, NDUFA4, NDUFA13, UCRC, and UQCRFS1) in blastocysts. The percentage of the sister blastomere pairs in which both cells developed to blastocysts (36.6 ± 5.3%) or both degenerated (46.3 ± 10.3%) were higher (P < 0.05) than that of the pairs in which one developed to blastocyst while the other degenerated (17.1 ± 7.8%). When both pairs developed to blastocysts, one blastocyst had more (P < 0.05) ICM and TE cells (8.2 ± 1.2 and 20.2 ± 2.1, respectively) than the other (5.2 ± 0.9 and 13.5 ± 1.1), although ICM:TE cell ratios were not significantly different. In conclusion, blastomere separation at the two-cell stage significantly increased blastocyst yield from IVP porcine embryos. This might be a useful approach for conservation of rare pig breeds, in which low numbers of embryos limited the success of embryo transfer.
Theriogenology 01/2011; 1(76(1)):88-96. · 1.96 Impact Factor
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ABSTRACT: We experimentally demonstrate parametric tunable dispersion compensation with a spectrally non-inverting tunable wavelength converter based on cascaded sum- and difference-frequency generation of PPLN waveguide. An error-free 43-Gbit/s NRZ-OOK transmission over 53.2-km SMF is successfully achieved with a low-power penalty.
Optical Communication (ECOC), 2010 36th European Conference and Exhibition on; 10/2010
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ABSTRACT: This paper proposes real-time sound source orientation estimation based on orientation-extended amplitude beamforming (OE-ABF). To recognize a sound source orientation (such as face orientation) is an important function for a robot who can achieve natural human-robot interaction because the function is required to distinguish the human target from a robot or another person. We developed a sound source orientation system using orientation-extended beamforming (OE-BF) and showed the system worked properly at least under a specific controlled environment. However, in practical use, this system does not work properly because the system doesn't take into account the differences between the supposed model in OE-BF and in practical situations. For example, the system model supposes that there is neither noise nor reverberation, however, this is not a realistic assumption. To solve this assumption mismatch problem, we propose sound source orientation estimation based on OE-ABF, and constructed a real-time sound source orientation estimation system with the proposed method using a 96 ch microphone array. Evaluation results of our proposed system show that the average error of estimated angles is lower than 5°, while the error of our previously reported system was greater than 20°. With this system, the robot is able to distinguish that the utterance target of a person standing 1 m in front is itself or another person standing 0.2 m to the left of the robot. This is valuable for human-robot interaction.
Intelligent Robots and Systems, 2009. IROS 2009. IEEE/RSJ International Conference on; 11/2009
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ABSTRACT: We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.
Theriogenology 10/2009; 73(2):147-56. · 1.96 Impact Factor
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ABSTRACT: It is anticipated that the utilization of spermatogonia through testicular xenografting will open new avenues for the conservation of male gametes. With the aim of establishing this new technique for genetic preservation of pigs, we used it in combination with intracytoplasmic sperm injection (ICSI). Testicular tissues derived from neonatal piglets, which contained seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 125 and 192 d after xenografting, sperm (morphologically similar to epididymal sperm) were recovered from 41 of the 65 host mice (63.1%). Testicular spermatozoa from adult boars were used as a positive control. A single spermatozoon was injected into an in vitro matured porcine oocyte, and the oocytes were electro-stimulated and cultured (graft-ICSI and testis-ICSI, respectively). Blastocyst rates in both ICSI groups (24.9% and 37.4%, respectively) were higher (P<0.05) than those without the injection procedure (parthenogenetic; 12.7%) and after injection of a small amount of injection buffer (sham; 13.0%). Rates of diploid blastocysts in both graft-ICSI and testis-ICSI groups (48.9% and 60.6%) were higher (P<0.05) than those in the parthenogenetic and sham groups (13.5% and 28.0%). Therefore, we demonstrated that porcine oocytes injected with xenogeneic sperm have in vitro developmental ability to the blastocyst stage.
Theriogenology 02/2009; 72(1):2-9. · 1.96 Impact Factor
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ABSTRACT: In pigs, the intracytoplasmic sperm injection (ICSI) procedure alone is insufficient to induce oocyte activation for embryonic development. Artificial activation can be accomplished, such as by electrical pulse-enhanced in vitro development to the blastocyst stage (Nakai et al. 2006). It is well known that the sperm factor (phospholipase Czeta; PLCzeta) in spermatozoa, which triggers oocyte activation, is diffused into ooplasm when sperm fuse with oocytes. Our previous study showed that the activation rate of porcine oocytes injected with one sonicated sperm head was significantly lower than that of oocytes injected with a whole spermatozoon or with 3 sonicated sperm heads (Nakai et al. IETS 2007). These results suggest that the sonication treatment per se may affect the quantity of PLCzeta in sperm. Furthermore, various pretreatments of sperm besides sonication have been conducted (e.g. removal of the sperm membrane) to increase the efficacy of ICSI. In this study, we investigated the effect of pretreatments (sonication, Triton X-100, and repeated cycles of freezing-thawing without cryoprotectant) on the quantity of PLCzeta in porcine sperm. Cryopreserved-thawed boar-ejaculated sperm were used for 3 experimental groups: (1) sperm were sonicated for 10 s in pig-fertilization medium (pig-FM; Suzuki et al. 2002; Soni group), (2) freezing-thawing was repeated 3 times in pig-FM without cryoprotectant (3-F/T group), or (3) sperm were incubated in pig-FM supplemented with 0.1 or 1% Triton X-100 at 37 degrees C for 1 min (0.1 and 1% Triton X-100 groups, respectively). Cryopreserved-thawed whole sperm without any treatment was used as a control. Results from staining with fluorescein diacetate and propidium iodide showed that almost all sperm were propidium iodide positive (dead sperm) immediately after the each treatment. In the control group, approximately 40% of sperm were fluorescein diacetate positive (live sperm) after thawing. The presence of PLCzeta (72 kDa) was examined by Western blotting using the antibody against the N-terminal 19-mer sequence of porcine PLCzeta (Kurokawa et al. 2005). A band corresponding to porcine PLCzeta was not detected in any treatment group in any culture period (from 0 to 135 min). In contrast, PLCzeta was detected in the control group and in all culture periods. These results strongly suggest that PLCzeta in porcine sperm was lost immediately after the pretreatments, such as by sonication, incubation with 0.1 or 1% Triton X-100, and repeated cycles of freezing-thawing. The decrease in PLCzeta protein by pretreatment may be one of the causes of incomplete activation of oocytes in porcine ICSI.
Reproduction Fertility and Development 01/2009; 21(1):232-233. · 2.11 Impact Factor
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ABSTRACT: In vitro production (IVP) including in vitro maturation (IVM) and fertilization (IVF) is now an important technology for obtaining live piglets. However, there are still two significant obstacles to the efficient production of viable porcine embryos: (1) polyspermy and (2) fertilization of oocytes arrested at the immature stage. These phenomena relate to production of embryos with abnormal ploidy (polyploidy). To avoid these problems, careful selection of mature oocytes for IVF, and regular monitoring of normal and abnormal fertilization (polyspermy and/or lack of male pronucleus formation) are very important. In our recent studies, however, we have confirmed that some oocytes with abnormal ploidy after polyspermy can develop into diploid embryos with potentially normal developmental ability. The mechanism by which such fertilized polyploid oocytes develop to a normal state during embryo development is still not well understood. Attempts to clarify this mechanism would hopefully reveal data that are very useful for not only IVP but also other technologies such as the production of transgenic or cloned animals using IVM oocytes, including other species, also for human reproductive manipulation. In this review, we focus on studies of normality of IVM oocytes and ploidy of IVP embryos, and try to suggest practical ways of solving the problems mentioned above in pigs.
Society of Reproduction and Fertility supplement 01/2009; 66:135-47.
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N Maedomari, K Kikuchi,
T Nagai,
M Fahrudin,
H Kaneko,
J Noguchi,
M Nakai,
M Ozawa,
T Somfai,
L V Nguyen,
J Ito,
N Kashiwazaki
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ABSTRACT: The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.
Reproduction in Domestic Animals 01/2009; 45(4):659-65. · 1.36 Impact Factor
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ABSTRACT: In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM-IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo-derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.
Reproduction in Domestic Animals 07/2008; 43 Suppl 2:401-6. · 1.36 Impact Factor
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ABSTRACT: Cysteine is considered to promote male pronuclear (MPN) formation in porcine through oocyte glutathione (GSH) synthesis (Yoshida et al. 1993 Biol. Reprod. 49, 89–94). The GSH has an important role in providing cells with a redox state and in acting to protect cells from toxic effects of oxidative damage (Meister et al. 1976 AM Rev. Biochem. 45, 559–604). However, such previous investigations were carried out under high O2 tension (20% O2) incubation conditions. Here we simply study IVM-IVF-IVC competence of porcine oocytes matured in IVM media supplemented with cysteine of different concentrations under low oxygen tension (5% O2). Cumulus–oocyte complexes (COCs) from prepubertal gilts were collected, matured, and fertilized in vitro according to Kikuchi et al. (2000 Biol. Reprod. 66, 1033–1041). COCs were cultured in IVM medium supplemented with 0 (Group 1; control), 0.05 (Group 2), 0.1 (Group 3), 0.2 (Group 4), and 0.6 mm (Group 5) cysteine under low oxygen tension. Nuclear maturation of oocytes, fertilization status, and number of cells in resultant embryos were assessed with orcein staining; also, the GSH content of IVM oocytes was measured by the method described by Ozawa et al. (2002 Reproduction 124, 683–689). Maturation rates of Groups 1–5 were 68.2 ± 3.2, 70.6 ± 7.7, 69.7 ± 15.9, 75.9 ± 7.7, and 68.8 ± 8.0%, respectively, indicating no difference in maturation competence among the groups (P > 0.05 by ANOVA). The rates of sperm penetration, MPN formation (95.9 ± 2.4, 100 ± 0, 92.8 ± 4.7, 94.0 ± 4.1, and 92.4 ± 2.7%, respectively), monospermy, and even blastocyst rates after 6 days of IVC were not different among the groups (P > 0.05 by ANOVA). Moreover, the cell numbers of blastomeres in blastocysts (38.68 ± 3.5, 40.1 ± 3.1, 37.5 ± 3.0, 36.2 ± 3.3, and 43.8 ± 4.0, respectively) were uniformly the same among the groups (P > 0.05 by ANOVA). However, GSH content of IVM oocytes increased significantly (P < 0.05 by ANOVA) as the concentration of cysteine increased (12.2 ± 0.6, 14 ± 0.8, 15.1 ± 0.5, 16.4 ± 0.4, and 16.4 ± 0.5 pmol/oocyte, respectively). The GSH level of oocytes in Group 1 (control) seems to be higher than that reported by Aberydeera et al. (1998 Biol. Reprod. 58, 213–218), who matured porcine oocytes under high O2 tension. This may reflect the effect of low O2 tension and explain the same developmental rate to the blastocyst stage as that of oocytes matured in the media supplemented with cysteine in this study. In conclusion, an addition of 0.05–0.6 mm cysteine during IVM, under 5% O2 tension, of porcine oocytes significantly increased intracellular GSH synthesis according to its concentration. However, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation, and subsequent embryonic development to the blastocyst stage. Thus, O2 tension during IVM of oocytes is suggested to be important for the in vitro production of porcine blastocysts.
Full text doi:10.1071/RDv20n1Ab247
Reproduction Fertility and Development 01/2008; 20(1):203. · 2.11 Impact Factor
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ABSTRACT: Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 ± 0.8% and 91.4 ± 3.6%, respectively) or between the 36E and 36U groups (98.2 ± 1.1% and 88.0 ± 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 ± 5.7% and 61.7 ± 7.8%, respectively) and total cell numbers (39.1 ± 2.1 and 31.7 ± 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 ± 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 ± 5.1%), the total cell number was not different (26.3 ± 5.5 and 25.3 ± 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 ± 6.3%) than did the 36-h embryos (28.2 ± 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 ± 2.6) were significantly higher than those of the 36-h embryos (26.2 ± 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 ± 7.0%) and greater cell numbers (40.6 ± 2.6) than those not showing a second division during this period (30– group; 46.8 ± 5.0% and 19.9 ± 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.
Reproduction Fertility and Development 01/2008; 20(1):188-189. · 2.11 Impact Factor
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ABSTRACT: It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0h group) were cultured for 24h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20h without supplements (second culture step; 44h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24h group) or partly (H-DO 24h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24h (DO 24h+CC group). The maturation rates of all the cumulus-removed groups (DO 0h, DO 24h, H-DO 24h and DO 24h+CC groups) were lower (34.3-45.0%) than that of the control group (64.5%; P<0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0h, DO 24h and DO 24h+CC groups) were lower (4.03-5.26pmol/oocyte) than that of the control group (9.60pmol/oocyte; P<0.05); however, the H-DO 24h group had an intermediate value (7.0pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4-59.3%) than that of the control group (89.4%; P<0.05), whereas the H-DO 24h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0-4.5%) than that of the control group (19.9%; P<0.05), and the H-DO 24h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation (P<0.01) and blastocyst formation (P<0.01), and also with the number of cells per blastocyst (P<0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.
Theriogenology 04/2007; 67(5):983-93. · 1.96 Impact Factor
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ABSTRACT: Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1-4) did not increase, even at 180 min (0.7-4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120-180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0-60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39. The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.
Zygote 02/2007; 15(1):15-24. · 1.17 Impact Factor
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ABSTRACT: To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4-5 wk of age) to postpubertal (49-56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21-26 wk of age. However, the peak disappeared after 31-32 wk of age. As bulls aged, especially after 31-32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27-34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21-26 wk of age and a single peak between 20 and 30 kDa after 31-32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin alpha and inhibin betaB antibodies and of a 31-kDa band cross-reacting with inhibin alpha and inhibin betaA antibodies. The 47-kDa band was recognized by the alpha, betaA, and betaB antibodies. Immunohistochemisty of the testis at each age showed that inhibin alpha subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.
Biology of Reproduction 06/2003; 68(5):1918-25. · 4.01 Impact Factor
