H. Kaneko

93 Production of live piglets by cryopreservation of in vitro-produced porcine zygotes

Successful cryopreservation of in vitro-produced porcine zygotes is reported in the present study. Follicular oocytes were collected from prepubertal gilts. They were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Ten or 23 h after IVF, the oocytes were centrifuged at 10 000g at 37°C for 20 min to permit visualization of pronuclei. Zygotes with two or three pronuclei were selected under stereomicroscope and used for solid surface vitrification (SSV). Briefly, after equilibration in 4% ethylene glycol (EG) for 15 min, zygotes were washed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 m trehalose), and then dropped with about 2 µL vitrification solution onto the dry surface of aluminum foil floating on the surface of liquid nitrogen (LN2). Microdroplets were transferred into cryotubes and stored in LN2. During warming, vitrified droplets were transferred in warming solution (0.4 m trehalose) at 37°C for 1 min, and then consecutively transferred for 1-min periods into 0.2 m, 0.1 m, or 0.05 m trehalose solutions. Survival of vitrified/warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CT), and untreated (control) zygotes were cultured in vitro for 6 days. There was no difference in developmental competence between control and CT zygotes in terms of cleavage rates (88.1% and 86.1%, respectively), blastocyst rates (23.2% and 20.8%, respectively), and blastocyst cell numbers (38.0 ± 2.0 and 41.2 ± 1.7, respectively). The rate of live zygotes after SSV and warming was similar to that of the control (93.4% and 100%, respectively). Cleavage rates (71.7% and 86.3%, respectively) and blastocyst rates (15.8% and 24.5%, respectively) of SSV were significantly reduced after vitrification compared to control (P < 0.05, ANOVA). Blastocyst cell numbers of SSV and control embryos were similar (41.2 ± 3.4 and 41.6 ± 3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. When embryo culture medium was supplemented with 1 µm of the antioxidant glutathione, development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of the control zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, 150 vitrified zygotes were transferred into a recipient, resulting in pregnancy and the production of five live piglets. These data demonstrate that a high rate of porcine zygotes could be successfully cryopreserved at the pronuclear stage, preserving their full developmental competence.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncans multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

350 EFFECT OF INDIRECT MAINTENANCE OF INTRACELLULAR cAMP IN CUMULUS–OOCYTE COMPLEXES USING 3-ISOBUTYL-1-METHYLXANTHINE ON GAP JUNCTIONAL COMMUNICATIONS AND OOCYTE MEIOTIC MATURATION

Oocyte and cumulus cells communicate through an extensive network of gap junctions (GJs), which permit the transfer of small molecules such as cAMP. Gonadotropin strongly enhances the intracellular cAMP concentration in cumulus cells, and induces oocyte meiotic resumption. Enhanced cAMP also triggers a reduction of GJ communications (GJCs) in cumulus-oocyte complexes (COCs), accompanied by cumulus expansion. Intracellular cAMP is modulated by both adenylate cyclase (AC) for synthesizing and phosphodiesterase (PDE) for degrading. Addition of AC to gonadotropin-free medium induces meiotic resumption of bovine oocytes without cumulus expansion, suggesting that maintenance of cAMP at a certain level in COCs may be crucial for either prolonged maintenance of GJCs or the timing of oocyte meiotic resumption. In the present study, we investigated the intracellular cAMP concentrations in porcine COCs or oocytes, and GJCs during in vitro oocyte maturation culture using PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Porcine COCs obtained from prepubertal gilts were cultured for 20 h (1st culture) using M199 containing 10% FCS (basic medium, BM group) with FSH (FSH group) or IBMX (IBMX group). Following this, the COCs were transferred into the basic medium containing FSH and LH, and cultured for another 24 h (2nd culture). At 6, 12, and 20 h of the 1st culture, intracellular cAMP in COCs or oocytes was measured. To determine GJCs in each COC, Lucifer Yellow fluorescent dye was microinjected into cumulus-enclosed oocytes at 6 or 12 h of the 1st culture, and the ability of dye transfer, which is related to the GJCs, from the oocyte to the surrounding cumulus cells was observed. At the end of the 1st culture, 30.8 ± 6.0% of the oocytes in the FSH group underwent germinal vesicle breakdown (GVBD), whereas only a few oocytes in the BM group (8.6 ± 2.4%) and the IBMX group (5.8 ± 3.0%) achieved GVBD (P < 0.05). In contrast, ratios of metaphase-II (M-II) stage oocytes at the end of the 2nd culture did not differ between the FSH group (75.7 ± 3.9%) and the IBMX group (68.2 ± 6.8%), although a few oocytes in BM group (10.1 ± 3.7%) reached the M-II stage (P < 0.05). Concentrations of cAMP in COCs and oocytes increased drastically in the FSH group compared to those of the BM and IBMX groups (P < 0.05). In addition, the concentration of cAMP in IBMX group oocytes was also higher than that in the BM group, with a significant difference detected at 20 h (P < 0.05). The GJCs in the FSH group were gradually closed, depending on the length of time in culture (54.9 ± 3.7% of COCs closed their GCJs at 12 h of the 1st culture). In contrast, in the IBMX group, only 23.0 ± 3.7% of COCs closed their GJCs at 12 h of the 1st culture, which was significantly different from that of the other two groups (P < 0.05). These results suggest that treatment with IBMX during the first half of IVM culture can induce subsequent meiotic resumption of porcine oocytes, and that a moderate increase of cAMP concentration in COCs or oocytes prolongs GJCs during the treatment.

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 µM (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 ± 2.3, 39.3 ± 1.7, and 42.5 ± 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 ± 1.6) and DPI-0.5 (32.7 ± 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 µM significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

35 RELATIONSHIP BETWEEN THE ONSET OF ENUCLEATION DURING MEIOTIC MATURATION OF RECIPIENT OOCYTES AND DEVELOPMENTAL ABILITY OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

In somatic cell nuclear transfer (SCNT), maturation promoting factor (MPF) is believed to be one of the factors involved with nuclear envelope breakdown and chromatin condensation of the transferred nucleus. Although MPF activity is high both in metaphase-I or -II oocytes (M-I and M-II, respectively), only M-II oocytes have been used exclusively as recipient cytoplasts in SCNT. In this study, we examined the effect of different onset of (1) enucleation of recipient oocytes at the M-I and M-II stages, and (2) fusion and activation of the couplets on their developmental ability to the blastocyst stage in pigs. The primary cultured cumulus cells were used as donor karyoplasts, and recipient cytoplasts were prepared by enucleation of in vitro-matured oocytes using gradient centrifugation in percoll solution. A karyoplast and a cytoplast were fused by 2 DC pulses of 1.5 kV cm-1 for 20 µs, and then the couplets were activated by 2 DC pulses of 0.8 kV cm-1 for 30 µs. The reconstructed embryos were cultured according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) except for the addition of 5% FCS to NCSU-37 during Days 2–7 (Day 0 is the day of SCNT) of embryo culture using the WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264). Some of the embryos were fixed at 1, 10, and 24 h after activation and examined for morphology of nuclei. After 30 h of IVM, oocytes (mainly at the M-I stage) were enucleated. Then the couplets were fused immediately (Group A) or at 48 h after the onset of IVM (Group B); activation was conducted at 48 h of IVM (Group A) or at 1 h after fusion (Group B). As a control group, oocytes were enucleated after 48 h of IVM and then the couplets were fused and activated. None of the embryos in Group B developed to the blastocyst stage. However, a few of the embryos [2/117 (1.7%)] in Group A developed to the blastocyst stage; however, the rate was significantly lower than that of the control group [10/112 (8.9%); chi-square; P = 0.03]. The rates of embryos undergoing premature chromosome condensation (PCC) in Group B at 1 h and 10 h after activation were significantly lower than those in Group A [1 h: 51/69 (73.9%) vs. 76/76 (100%); 10 h: 24/76 (31.6%) vs. 45/91 (49.5%), respectively); some of them had pseudo-pronuclei. By 24 h after activation there were no detectable differences in the rates of cleavage [2/70 (2.9%) vs. 2/61 (3.3%)]; however, the rates were significantly lower than that of the control group [23/90 (25.6%); chi-square; P < 0.05]. These results suggest that MPF activity might be changed in oocytes without nucleus during the maturation culture. Thus, a specific nucleus-associated factor(s) that may present in the cytoplasm seems to be essential for the successful remodeling of the transferred nucleus and the development of SCNT embryos to the blastocyst stage.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 µg/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 µs, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 µs at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H2O2 level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H2O2. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 µM GSH (29.6 ± 2.7% or 30.4 ± 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 ± 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 ± 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 ± 2.1 to 49.2 ± 2.1) and lowered ratios of apoptotic cells (6.2 ± 0.6% to 7.0 ± 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 ± 2.0, apoptosis rate = 11.1 ± 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.

318 INHIBITION OF FIRST POLAR BODY EXTRUSION BY CYTOCHALASIN B DURING IN VITRO MATURATION OF PORCINE OOCYTES LEADS TO RE-ARRANGEMENT OF THE SEGREGATED CHROMOSOMES AND IMPROVEMENT IN THE QUALITY OF THE PARTHENOGENETIC BLASTOCYSTS

Diploid parthenotes are usually obtained by the inhibition of second polar body (PB2) extrusion after activation of metaphase II (MII) oocytes. However, diploid embryos can be generated by the inhibition of the first polar body (PB1) extrusion as well, using cytochalasin B (CB) during in vitro maturation prior the activation procedure. A higher percentage of mouse embryos generated by the activation of MII oocytes and the inhibition of PB2 extrusion were proven to be homozygous than for parthenotes obtained by the latter method (Kubiak et al. 1991 Development 111, 763-769). The aim of the present study was to examine if such difference has any effect on the development of parthenogenetic embryos in vitro. Nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to CB during in vitro maturation (IVM) was investigated in the present study. The tendency of nuclear maturation was similar in oocytes matured in the presence of 1 ¼g/mL CB (IVM-CB group) and control oocytes matured without CB after 37 h of IVM; at this time the frequency of oocytes that had reached/or passed through anaphase-I stage did not differ significantly (P < 0.05) between the IVM-CB and the control groups (61.3% and 69.9%, respectively), however, no polar body extrusion was observed in the IVM-CB group and the two lumps of homologue chromosomes remained in the oocyte and turned into two irregular sets of condensed chromosomes. By 41 h of IVM, the double sets of chromosomes re-united in 89.5% of IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached metaphase-II stage (MII) by this time. When IVM-CB oocytes were electrically (1.5 kV/cm for 100 ¼s) activated and subsequently cultured without CB, 39% of the oocytes extruded a polar body (PB) and 82.9% of them had a female pronucleus. When those oocytes with PB were cultured, the blastocyst rate of the cleaved embryos did not differ (P < 0.05) from those of the control that were stimulated at MII and subsequently treated with CB (43.3% and 48.2%, respectively). The number of blastomeres in Day 6 blastocysts was significantly higher (P < 0.05) in the IVM-CB derived embryos than in those in the control group (47.8 and 40.7, respectively); moreover, the ratio of dead blastomeres (dead cells : live cells) was higher (P < 0.05) in the control than in the IVM-CB blastocysts (0.047 and 0.031, respectively). A possible explanation for this result might be a lower frequency of homozygous genes in IVM-CB parthenotes, in which segregation of sister chromatids were promoted instead of segregation of homologous chromosomes to obtain diploid embryos. In such embryos the expression of recessive lethal, sublethal and subvital genes might have a lower probability. This work was supported by the Japanese-Hungarian bilateral scientific and technological cooperation (TET JAP-11/02).