T Somfai

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

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Publications (108)209.97 Total impact

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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. · 2.58 Impact Factor
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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. · 2.58 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. · 2.58 Impact Factor
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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. · 2.58 Impact Factor
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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.
    Theriogenology 11/2014; · 2.08 Impact Factor
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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
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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; · 1.04 Impact Factor
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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 01/2014; 9(5):e97731. · 3.53 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 12/2013; · 2.81 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. · 2.58 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. · 2.58 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; · 1.76 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; · 2.14 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; · 1.76 Impact Factor
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    ABSTRACT: Because of recent advancements in reproductive technology, oocytes have an increasingly enriched value as a unique cell population to produce offspring. The growing oocytes in the ovary are an immediate potential source to serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were cultured on Millicell membrane inserts with culture medium that was supplemented with 4% polyvinylpyrrolidone (molecular weight: 360000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon inducing oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite-I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from the in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.
    Biology of Reproduction 07/2013; · 4.03 Impact Factor
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    ABSTRACT: 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.
    Journal of Reproduction and Development 05/2013; · 1.76 Impact Factor
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    ABSTRACT: The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.
    Animal Science Journal 05/2013; · 1.04 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 01/2013; 8(7):e70989. · 3.53 Impact Factor
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    ABSTRACT: The objective was to determine the effects of adding L-carnitine (an enhancer of lipid metabolism) during IVM, on cryotolerance and developmental competence of bovine oocytes. Oocytes matured in the absence (control) or presence (0.6 mg/mL) of L-carnitine were subjected to IVF and embryo culture after Cryotop vitrification or nonvitrification at the metaphase stage of the second meiotic cell division. Cleavage and blastocyst formation rates, and inner cell mass and trophectoderm cell numbers were determined. Also, ATP content in IVM oocytes was measured and intracellular lipid droplets were observed (Nile red staining and confocal microscopy). L-carnitine had no significant effect on the rate of matured oocytes. Vitrification reduced (P < 0.05) mean (±SEM) rates of live oocytes both in control (80.6 ± 1.9%) and L-carnitine groups (82.7 ± 5.1%) compared with nonvitrified oocytes (100%). After IVF, cleavage rates of vitrified control and L-carnitine groups (56.5 ± 3.9% and 62.8 ± 5.1%, respectively) were significantly lower than those in nonvitrified control and L-carnitine groups (83.9 ± 4.2% and 84.3 ± 1.3%). After vitrification, blastocyst formation rate in the L-carnitine group (54.4 ± 5.2%) was significantly higher compared with the control (34.9 ± 4.4%), and did not significantly differ from those in nonvitrified control and L-carnitine groups (52.1 ± 4.2% and 52.8 ± 3.0%). The numbers and ratio of inner cell mass and trophectoderm cells in blastocysts did not differ significantly among groups. The ATP content in L-carnitine-treated oocytes tended to be higher compared with the control. Vitrification did not reduce ATP content in oocytes, irrespective of L-carnitine treatment. Treatment with L-carnitine dislocated lipid droplets from the peripheral area to the inner cytoplasm. In conclusion, L-carnitine supplementation during IVM redistributed lipid droplets in oocytes; if they survived vitrification, their developmental competence was similar to that of nonvitrified oocytes.
    Theriogenology 12/2012; · 2.08 Impact Factor
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    ABSTRACT: Abstract Although telomeres are elongated during morula-to-blastocyst transition in cloned embryos, it is still unknown whether donor cell types have any effect on this elongation. In the present study, we examined the changes of telomere length during morula-to-blastocyst transition in cloned porcine embryos using different types of donor cells. Porcine embryonic stem-like cells (pESLCs), porcine cumulus cells (PCs), and porcine embryonic fibroblasts at passages 7 and 10 (PEF7s and PEF10s, respectively) were used as donor cells. Telomere lengths of pESLCs (35.8±1.5 kb), PCs (24.4±0.5 kb), PEF7s (18.7±0.6 kb), and PEF10s (17.2±0.1 kb) were significantly different. In contrast, telomere length in morulae derived from pESLCs (18.2±0.3 kb), PC (17.8±0.7 kb), PEF7 (18.5±0.3 kb), and PEF10 (18.4±0.4 kb) did not differ significantly. Likewise, telomeres in blastocysts derived from pESLCs (22.3±1.5 kb), PCs (23.5±2.6 kb), PEF7s (20.2±1.0 kb), and PEF10s (20.9±1.0 kb) had similar lengths. However, telomeres in blastocysts were significant longer (p<0.05) compared with morulae in each group. Relative telomerase activities of morulae derived from pESLCs (4.2±0.4), PCs (4.0±0.5), PEF7s (5.1±0.4), and PEF10s (4.9±0.4) were significantly lower (p<0.01) than those of blastocysts derived from pESLCs (8.2±1.1), PCs (8.6±0.6), PEF7s (12.5±2.9), and PEF10s (8.3±1.1). In conclusion, the telomere elongation in cloned pig embryos that occurred during morula-to-blastocyst transition may be related to the rise of telomerase activity. The telomere elongation may also be independent of the type and telomere length of the donor cell.
    Cellular reprogramming. 12/2012; 14(6):514-9.

Publication Stats

572 Citations
209.97 Total Impact Points

Institutions

  • 2006–2014
    • National Institute of Livestock and Grassland Science
      Ibaragi, Ōsaka, Japan
  • 2013
    • University of Tsukuba
      Tsukuba, Ibaraki, Japan
    • Research Institute for Animal Breeding and Nutrition, Hungary
      Budapeŝto, Budapest, Hungary
  • 2009–2012
    • National Livestock Breeding Center
      Hukusima, Fukushima, Japan
    • Suranaree University of Technology
      • School of Biotechnology
      Khorat, Nakhon Ratchasima, Thailand
  • 2003–2010
    • National Institute of Agrobiological Sciences
      • Division of Animal Sciences
      Tsukuba, Ibaraki-ken, Japan
  • 2005
    • Prime Tech Ltd.
      牛久, Ibaraki, Japan
  • 2002
    • University of West Hungary, Sopron
      Scarabantia, Győr-Moson-Sopron, Hungary