T Somfai

National Institute of Livestock and Grassland Science, Ibaragi, Ōsaka, Japan

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Publications (114)218.24 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG=1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO=1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5-15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.
    Journal of Reproduction and Development 09/2015; DOI:10.1262/jrd.2015-089 · 1.52 Impact Factor
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    ABSTRACT: The aim of the present study was to optimize the temperature for the temporal storage of matured bovine oocytes. In vitro-matured bovine oocytes were preserved in HEPES-buffered TCM199 medium supplemented with 10% newborn calf serum at different temperatures (4 °C, 15 °C, 25 °C, and 38.5 °C) for 20 hours. Embryo development and blastocyst quality after in vitro fertilization, cytoplasmic ATP and glutathione levels in oocytes, and the frequency of apoptotic oocytes were compared among storage groups and a control group without storage. Among the storage groups, those at 25 °C and 38.5 °C showed the highest rates of blastocyst development (19.3% and 24.5%, respectively) compared with those stored at 4 °C and 15 °C (8.5% and 14.9%, respectively); however, blastocyst formation rates in all storage groups were lower than that in the control group (39.8%; P < 0.05). Storage at 38.5 °C and 15 °C was associated with reduced cell numbers in resultant blastocysts compared with the control and the 25 °C storage groups. Storage at 4 °C reduced metabolic activity of oocytes characterized by their lower ATP levels compared with the other groups. Storage for 20 hours significantly reduced the glutathione content in oocytes in all groups in a similar manner, irrespective of the temperature. Storage at 4 °C or 15 °C but not at 25 °C and 38.5 °C significantly increased the percentage of apoptotic oocytes compared with the control group. In conclusion, 25 °C was found to be the most suitable temperature for the temporal storage of matured bovine oocytes regarding both the developmental competence of oocytes and the quality of resultant blastocysts.
    Theriogenology 09/2015; DOI:10.1016/j.theriogenology.2015.09.033 · 1.80 Impact Factor
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    ABSTRACT: Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system. © 2015 Japanese Society of Animal Science.
    Animal Science Journal 08/2015; DOI:10.1111/asj.12459 · 0.96 Impact Factor
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    ABSTRACT: Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick-Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non-stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H2 O2 levels at the metaphase-II stage and intracellular Ca(2+) levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re-distribution in non-stimulated OPU-derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non-stimulated OPU in terms of ATP content, cytoplasmic H2 O2 levels, base Ca(2+) levels and the frequencies and amplitudes of Ca(2+) oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence. © 2015 Japanese Society of Animal Science.
    Animal Science Journal 07/2015; DOI:10.1111/asj.12387 · 0.96 Impact Factor
  • S Matoba · T Somfai · T Nagai · M Geshi ·
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    ABSTRACT: Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unitmL(-1) FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A-C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25mgmL(-1) linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970-978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P>0.05) in total blastocyst formation rates on Day 8 (Day 0=IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P<0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P<0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.
    Reproduction Fertility and Development 12/2014; 27(1):208. DOI:10.1071/RDv27n1Ab237 · 2.40 Impact Factor
  • T Nagai · T Somfai · N T Men · H Kabeko · J Noguchi · K Kikuchi ·
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    ABSTRACT: We investigated the effects of collection season and storage duration of vitrified porcine oocytes in liquid nitrogen (LN2) on their survival and maturation ability after warming. A total of 3338 cumulus-enclosed oocytes were vitrified using solid surface vitrification, preserved, and warmed according to previous report (Somfai et al. 2014 PLoS One 9, e97731) in 26 occasions between October 2012 and March 2014. Vitrified oocytes were stored in LN2 for various durations from 0 (vitrified but without storage) to 243 days. The date of preservation and length of storage (days) of vitrified oocytes in LN2 were recorded. Warming of vitrified oocytes was conducted on a hotplate set at 42°C. After warming, oocytes were subjected to in vitro maturation according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033-1041). Then oocytes were denuded and their live/dead status and nuclear maturation were assessed under stereo microscope based on their morphology and the presence of the first polar body. After linear regression analysis, it was found that there was no correlation between the duration of storage of vitrified oocytes in LN2 for up to 243 days and their survival rate after warming (R=0.254; P=0.210) or the maturation rate of surviving oocytes (R=0.147; P=0.471). Vitrification during spring (March 1-May 31) resulted in significantly higher rates of survived oocytes compared with vitrification during winter (December 1-February 28; 86.9 and 73.1%, respectively; P<0.05), whereas the mean survival rates of oocytes vitrified during summer (June 1-August 31; 79.0%) and autumn (September 1-November 31; 81.9%) did not differ significantly from those of other seasons (ANOVA). After in vitro maturation, nuclear maturation of surviving oocytes did not differ significantly among oocytes vitrified at different seasons (ranging between 59.1 and 67.8%). The results indicate that the oocyte collection season affects survival of vitrified oocytes, whereas storage duration in LN2 does not affect this parameter. Furthermore, nuclear maturation of oocytes that survive after vitrification and warming is not affected by their collection season and storage length.
    Reproduction Fertility and Development 12/2014; 27(1):123-4. DOI:10.1071/RDv27n1Ab61 · 2.40 Impact Factor
  • T Somfai · N T Men · H Kaneko · J Noguchi · S Haraguchi · T Nagai · K Kikuchi ·
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    ABSTRACT: Cryotop and solid surface vitrification are frequently used methods for the cryopreservation of porcine oocytes. These methods differ not only in the vitrification carrier but also in the cryoprotectant (CPA) treatment including the type of sugar, permeable CPA (pCPA) combinations, and the equilibration regimen. This study compared the distinct points of CPA treatment of these 2 methods to determine the optimum CPA treatment for the solid surface vitrification of immature porcine oocytes. We vitrified and warmed follicular cumulus-oocyte complexes by our method (Somfai et al. 2014 PLoS One 9, e97731). In each experiment, the vitrification solution consisted of 50mgmL(-1) polyvinyl pyrrolidone, 0.3M of the actual sugar, and 35% [v/v] in total of the actual pCPA combination (depending on the experiment). After warming, the cumulus-oocyte complexes were subjected to in vitro maturation, IVF, and embryo culture (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Oocyte survival was assessed after IVF by morphological evaluation, and live oocytes were subjected to in vitro embryo culture. Cleavage and blastocyst rates were calculated from cultured oocytes on Day 2 (Day 0=IVF) and Day 6, respectively. Each experiment was replicated at least 3 times. Results were analysed by ANOVA. In Experiment 1, we compared trehalose (n=416) and sucrose (n=440) as supplementations during vitrification and warming (0.3M and 0.4M of each, respectively). There was no significant difference between oocytes vitrified with trehalose or sucrose in terms of survival, cleavage, and blastocyst development (83.2% v. 80.3%, 39.7% v. 42.4%, and 3.6% v. 5.9%, respectively). Thus, vitrification and warming media were supplemented with sucrose thereafter. In Experiment 2, we compared 1:1 combinations of ethylene glycol with propylene glycol (EG+PG group, n=452) and ethylene glycol with dimethyl sulfoxide (EG+DMSO group, n=465) used as pCPA for equilibration (4% [v/v] pCPA in total for 15min) and vitrification (35% [v/v] pCPA in total for 30s). Oocyte survival rate was higher (P<0.05) in the EG+PG group compared with the EG+DMSO group (73.8% v. 51.1%, respectively); however, cleavage and blastocyst development rates of surviving oocytes were not significantly different between the 2 groups (30.5% v. 44.5% and 4.1% v. 6.3%, respectively). In Experiment 3, we compared an equilibration treatment in 4% [v/v] of EG+PG for 13 to 15min (regimen A, n=368) with an equilibration in 15% [v/v] of EG+PG for 5 to 7min (regimen B, n=363) for oocyte vitrification. Survival, cleavage, and blastocyst development rates were higher (P<0.01) for oocytes vitrified using regimen A compared with those vitrified using regimen B (82.5% v. 22.7%, 24.0% v. 7.7%, and 3.2% v. 0%, respectively). In conclusion, trehalose and sucrose are equally effective during vitrification and warming, the combination of EG+PG as pCPA is superior to EG+DMSO, and equilibration in 4% pCPA for 13 to 15min is superior to that in 15% pCPA for 5 to 7min for the vitrification of immature porcine oocytes.
    Reproduction Fertility and Development 12/2014; 27(1):124. DOI:10.1071/RDv27n1Ab62 · 2.40 Impact Factor
  • R Appeltant · T Somfai · M Nakai · S Bodo · D Maes · K Kikuchi · A Van Soom ·
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    ABSTRACT: Recent research has revealed that oocyte-secreted factors (OSF) affect cumulus expansion and play important roles during maturation and embryo development of mammalian oocytes. The use of denuded oocytes (DO) as supplements during in vitro maturation (IVM) in a nondefined medium improved developmental competence of cumulus-enclosed porcine oocytes (COC; Gomez et al. 2012 Zygote 20, 135-145). We investigated the effect of DO on cumulus expansion and nuclear maturation of COC in pigs during IVM using a defined medium. If the DO exert a positive influence on IVM, the defined medium can then be analysed for the presence of OSF. Immature COC were collected in the slaughterhouse from prepubertal gilts. To obtain DO, some COC were completely denuded by pipetting through a narrow-bore glass pipette. The COC used as a source for DO fulfilled the same morphological criteria as the COC used for IVM. The IVM medium was porcine oocyte medium (POM; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208-213) with hormone supplementations applied only during the first 20h of the IVM period. The COC were fixed to the bottom of 35-mm plastic Petri dishes in 3×3 grids by Cell-Tak (BD Bioscience, Bedford, MA, USA) in 100-µL droplets POM covered by paraffin oil. Culture droplets (each including 1 COC grid) were supplemented with (DO+ group, n=179) or without 16 DO (DO- group, n=143). After 20h of IVM, the medium was replaced with a preincubated hormone-free POM and oocytes were cultured for an additional 28h. At 0, 20, and 48h of IVM, images of each grid were taken at the same magnification. The size of each COC was measured as a 2-dimensional area in pixels by analysing images with ImageJ software. Relative cumulus expansion was calculated at 20 and 48h of IVM on the basis of the initial COC size at 0h, which was assigned as 1. At 48h of IVM, the COC were denuded and examined for oocyte maturation by orcein staining. The experiment was replicated 5 times. Cumulus expansion ratios at 20 and 48h of IVM were compared between the DO+ and DO- groups by ANOVA. Maturation rates were compared between the DO+ and DO- groups by binary logistic regression. No difference in cumulus expansion between DO- and DO+ could be observed at 20h (1.83±0.04 and 1.75±0.03, respectively) and 48h (1.41±0.03 and 1.47±0.02, respectively) of IVM. Nuclear maturation rates of COC in DO- and DO+ groups did not differ significantly (39.0±5.4 and 32.9±8.8%, respectively). In conclusion, addition of DO to the defined IVM medium did not affect the cumulus expansion and oocyte maturation of follicular porcine COC. Further research is needed to assess the effects of DO during IVM on subsequent fertilization. If DO prove to be beneficial for fertilization, the nature of the OSF will be investigated.
    Reproduction Fertility and Development 12/2014; 27(1):237. DOI:10.1071/RDv27n1Ab296 · 2.40 Impact Factor
  • R Appeltant · T Somfai · M Nakai · S Bodó · D Maes · K Kikuchi · A Van Soom ·
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    ABSTRACT: The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development. Copyright © 2014 Elsevier Inc. All rights reserved. Full text: http://authors.elsevier.com/a/1QSA0,28Lgjb4W
    Theriogenology 11/2014; DOI:10.1016/j.theriogenology.2014.10.026 · 1.80 Impact Factor
  • Nguyen Men · K.Kikuchi · M. Nakai · H. Kaneko · J. Noguchi · T. Somfai · A. Fukuda · A.Tajima ·
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    ABSTRACT: Mammalian sperm preservation by freeze drying has been considered as a safe and cheap approach relative to cryopreservation in recent years. In previous study, we reported that boar sperm freeze dried in a medium containing 15 mM trehalose maintain DNA integrity better than in those of without trehalose but there were no significant differences in fertilization and blastocyst formation rate. Porcine oocytes are characterized by a dark, granulated ooplasm due to its high lipid content and thus, considered more difficult to manipulate than other species (human, mice). Centrifugation (CF) to stratify cytoplasm has been conducted to facilitate the observation of sperm release in ooplasm (Wall et al, 1985). Our hypothesis is ‘the redistribution of cytoplasm after CF may facilitate the interaction between sperm and oocyte, thus, increase fertilization’. In experiment 1, mature oocytes were centrifuged at 1000rpm, 20min, 37oC to investigate the relative distance of chromosome-spindle complex (CSC) to polar body (PB) and compared with before CF. The objective of this experiment is to investigate the relative distance of CSC relative to PB before and after CF. Three types of location were classified (A, B, C) and 3 distinctive layers were observed as described by Fahrudin et al, 2007 (No organelle, Mitochondria and Lipid). CSC migrated far the way to PB (type B) was 2-fold higher in CF-oocytes compared with before CF as a result of CF. In experiment 2, mature oocytes were CF before injection, then FD sperm from Trehalose 0 mM were injected into 3 different layers of CF-oocytes. The effect of oocyte CF before injection was assessed. One hour after injection, electric activation (EA) was applied to injected oocytes. The oocytes were cultured in vitro in IVC Pyr/Lac for 9h and then fixed and stained to examine fertilization. There were no differences in normal or abnormal fertilization among all groups compared with control (oocyte without CF). In experiment 3, FD sperm from Trehalose 15 mM group were used. One hour after injection, the oocytes were subject to CF combined with or without EA (CF+EA+; CF+EA-) or without CF combined with or without EA (CF-EA+; CF-EA-) and cultured in vitro in IVC Pyr/Lac for 9h and then fixed and stained to examine fertilization status. There was no effect of CF on any parameters of fertilization. However, normal fertilization rate was significantly improved in groups with EA applied (P<0.05, one-way ANOVA). These results confirm the importance of additional electric stimulation to activate the pig ICSI-oocytes. Notably, the percentage of MIII arrested-oocytes was significantly higher in the groups without EA suggest that mechanical injection procedure is not sufficient to induce oocyte activation to proceed to PN stage
    Ag-ESD symposium, Tsukuba, Japan; 11/2014

  • 09/2014; DOI:10.1530/repabs.1.P157
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    ABSTRACT: 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.
    PLoS ONE 05/2014; 9(5):e97731. DOI:10.1371/journal.pone.0097731 · 3.23 Impact Factor
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    ABSTRACT: We produced recombinant porcine leukemia inhibitory factor (pLIF) and examined its effect on in vitro maturation (IVM) of porcine oocytes and their developmental competence after in vitro fertilization. Porcine cumulus-oocyte complexes (COCs) were matured in a medium supplemented with pLIF during the first 22 h, last 22 h, or entire 44 h duration of IVM. Oocytes in all groups tended to show enhanced nuclear maturation rates by the metaphase II (MII) stage (76.1%, 82.1%, and 86.6%, respectively) compared to the without-pLIF treatment group (69.6%, control). A significant increase in MII rate (P < 0.05) and obvious induction of cumulus expansion were observed over the whole time span (44 h) in the IVM group. When cumulus cells were removed at 22 h and denuded oocytes were further cultured, pLIF showed no effect on maturation rate. Oocytes matured in pLIF-supplemented medium showed a tendency for more rapid blastocyst development (21.1% vs. 16.2%, P = 0.0715). Examination of transcripts and proteins of the LIF signaling pathway in COCs revealed that LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) are present in both cumulus cells and oocytes. The amount of phosphorylated STAT3 (p-STAT3) markedly increased in both cumulus cells and oocytes cultured in pLIF-supplemented media, although oocyte p-STAT3 disappeared after 44 h of IVM. These results suggest that the LIF/STAT3 pathway is functional during IVM of porcine oocytes, and supplementing pLIF in the IVM medium can improve oocyte maturation by activating this pathway. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 03/2014; 81(3). DOI:10.1002/mrd.22289 · 2.53 Impact Factor
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    ABSTRACT: In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P < 0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P < 0.05) than that of large blastomeres and blastomeres from evenly cleaved 2-cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2-cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.
    Animal Science Journal 02/2014; 85(5). DOI:10.1111/asj.12183 · 0.96 Impact Factor
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    ABSTRACT: Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5μgmL(-1) of cytochalasin B (CB) for 30min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5μgmL(-1) CB for 13 to 15min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3M trehalose in BM) and then dropped with 2μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10-25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5mL of warming solution (0.4M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05M trehalose at 38°C. The COC were matured in vitro for 44h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208-213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208-213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0=the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P<0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world.
    Reproduction Fertility and Development 12/2013; 26(1):136. DOI:10.1071/RDv26n1Ab44 · 2.40 Impact Factor
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    ABSTRACT: A variety of growth factors and cytokines that are present in follicular fluid provide oocytes with a suitable environment for their maturation. One such cytokine is leukemia inhibitory factor (LIF). Although LIF-supplemented medium enhances embryo development in human, mouse, and bovine, studies investigating the effects of LIF on in vitro maturation (IVM) and subsequent embryo development are inconclusive. Additionally, the underlying mechanisms of LIF in oocyte maturation and embryo development after IVF have not been studied yet. In the present study, we examined the effect of recombinant porcine LIF (pLIF), produced in our laboratory, on porcine oocyte maturation and the mechanism of how LIF involves in oocyte maturation process at molecular level. The biological activity of pLIF was evaluated by sustenance of mouse embryonic stem (ES) cells with an undifferentiating state in ES medium supplemented with pLIF, and the final concentration (1:200, equivalent to 1000UmL(-1) of mouse LIF) was determined by serial dilution. Porcine cumulus-oocyte complexes (COC) were cultured in modified NCSU-37 medium supplemented with pLIF during the first 22h [pLIF (+, -)], the latter 22h [pLIF (-, +)], or whole 44h [pLIF (+, +)] of IVM and the proportion of metaphase II (M-II) stage oocytes was observed. Oocyte maturation was enhanced in each group by supplementation with pLIF [pLIF (+, -): 76.1%, n=138; pLIF (-, +): 82.1%, n=140; pLIF (+, +): 86.6%, n=127], when compared with control [pLIF (-, -): 69.6%, n=112], in which a significant increase of M-II rate (P<0.05 by ANOVA) and cumulus expansion were observed in the pLIF (+, +) group. The effect of pLIF was only seen for COC but not for denuded oocytes. When oocytes were subjected to IVF (Kikuchi et al. 2002), those matured in pLIF (+, +)-supplemented medium demonstrated higher blastocyst developmental rates (21.1% v. 16.2%; P=0.07) with increased cell numbers (50.2 cells v. 45.0 cells; P=0.12) compared with pLIF (-, -) on Day 6 of embryo culture (IVF=0). Examination of transcripts and proteins of the LIF signalling pathway revealed that mRNA and protein levels of LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) were similar in both pLIF (-, -) and pLIF (+, +) samples. However, notable phosphorylation of STAT3 was observed in the pLIF (+, +) sample. These results suggest that the LIF/STAT3-pathway is functional during oocyte maturation in pigs. Therefore, supplementation of maturation medium with pLIF could improve the developmental competence of oocytes by activation of this pathway.
    Reproduction Fertility and Development 12/2013; 26(1):192. DOI:10.1071/RDv26n1Ab157 · 2.40 Impact Factor
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    Yuji Hirao · Tamas Somfai · Kenji Naruse ·
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    ABSTRACT: Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation.
    Journal of Reproduction and Development 10/2013; 60(1). DOI:10.1262/jrd.2013-089 · 1.52 Impact Factor
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    ABSTRACT: 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.
    Cryobiology 08/2013; 67(3). DOI:10.1016/j.cryobiol.2013.08.009 · 1.59 Impact Factor
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    ABSTRACT: 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.
    Journal of Reproduction and Development 08/2013; 59(6). DOI:10.1262/jrd.2013-042 · 1.52 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 07/2013; 8(7):e70989. DOI:10.1371/journal.pone.0070989 · 3.23 Impact Factor

Publication Stats

895 Citations
218.24 Total Impact Points


  • 2007-2015
    • National Institute of Livestock and Grassland Science
      Ibaragi, Ōsaka, Japan
    • Azabu University
      Sagamihara, Kanagawa, Japan
  • 2011
    • National Agriculture and Food Research Organization
      Tsukuba, Ibaraki, Japan
  • 2009-2011
    • National Livestock Breeding Center
      Hukusima, Fukushima, Japan
  • 2004-2010
    • National Institute of Agrobiological Sciences
      • Division of Animal Sciences
      Tsukuba, Ibaraki, Japan
  • 2008
    • The Graduate University for Advanced Studies
      Миура, Kanagawa, Japan
  • 2003
    • Tohoku University
      • Graduate School of Agricultural Science
      Sendai-shi, Miyagi, Japan
  • 2002
    • Research Institute for Animal Breeding and Nutrition, Hungary
      Budapeŝto, Budapest, Hungary
    • University of West Hungary, Sopron
      Scarabantia, Győr-Moson-Sopron, Hungary