Blastocyst Total Cell Number Research Articles

404. Repercussions of a transient decrease in pH on embryo viability and subsequent fetal development

Changes in the environment to which the preimplantation embryo is exposed can significantly influence fetal outcomes, indicative of ‘embryo programming'. Although many previous in vitro stress models have demonstrated programming changes using high dose stress effects, it is possible to induce similar effects with a physiologically relevant stress model. This study investigates the effect of a subtle transient pH change, during the first cleavage division, on blastocyst viability and fetal outcomes after embryo transfer. Zygotes from F1 hybrid mice (C57BL6xCBA F1) were cultured to the 2-cell stage (19h) in either control G1 medium or in G1 containing a weak acid, 2 mM DMO (5,5-Dimethyl-2,4-oxazolidinedione), then cultured to the blastocyst stage in control media G1/G2 (72h). Exposure to DMO induced a decrease in intracellular pH from 7.25 (control) to 7.10 (DMO). At the blastocyst stage, inner cell mass (ICM) cell number and cellular apoptosis were assessed, or embryos were transferred to pseudopregnant recipients to assess implantation and fetal outcomes. Differences were assessed using Student's t-test or generalised linear modelling followed by post-hoc tests. Exposure to DMO during the first cleavage division significantly reduced total blastocyst cell number from 83.0 ± 6.4 to 63.6 ± 3.8 (P < 0.05), reduced ICM number from 30.6 ± 3.6 to 20.2 ± 1.8 (P < 0.05) and significantly increased the apoptotic cell index from 1.9% to 3.2% (for control verses DMO embryos respectively) (P < 0.05). Blastocyst development was unchanged. Exposure to DMO during the first cleavage division did not alter implantation rates however fetal weight was decreased from 1058.9 mg ± 25.2 (control) to 949.1 mg ± 26.7 (DMO) (P < 0.05) and crown–rump length decreased from 21.9 mm ± 0.4 (control) to 20.6 mm ± 0.5 (DMO) (P < 0.05). In conclusion, this study demonstrates that a transient reduction in pH of only 0.15 units during early preimplantation embryo development significantly reduces resultant blastocyst viability and perturbs fetal growth, indicative of altered embryo programming. The mechanism behind this permanent change however is currently unknown.

Effects of culture conditions and nuclear transfer protocols on blastocyst formation and mRNA expression in pre-implantation porcine embryos

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis ( BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P < 0.05), number of inner cell mass cells (13.3 ± 4.3 vs. 7.6 ± 2.2, P < 0.05), and total cells (57.9 ± 19.5 vs. 36.3 ± 8.2, P < 0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0 mM calcium in fusion/activation medium (1.0C), and 7.5 μg/mL cytochalasin B treatment (0.1C&CB) yielded higher rates ( P < 0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1 mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased ( P < 0.05) in 1.0C (77.4 ± 28.9) compared to the control (58.5 ± 22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes ( BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice

Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as growth differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.

Addition of Macromolecules to PZM-3 Culture Medium on the Development and Hatching of In vitro Porcine Embryos

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir- derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA→(day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA→PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at 37.8±16.1 than the other groups at 16.8±4.4 - 30.1±10.9; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts. (Key Words : Porcine Embryos, Macromolecule, Blastocysts, Cell Number)

Role of nonessential amino acids on porcine embryos produced by parthenogenesis or somatic cell nuclear transfer

Amino acids play a multitude of roles during early embryonic development and have been demonstrated to facilitate improved development of in vivo or in vitro fertilized and parthenogenetic embryos in several species. However, review of emerging literatures, shows that culture milieu of cloned embryos might be different from those of in vitro fertilized embryos. This study therefore, evaluated the effect of nonessential amino acids (NEAA) on yield and quality of porcine embryos produced by somatic cell nuclear transfer (SCNT) and compared them with parthenogenetic embryos as control. Analysis showed that, supplementation of NEAA to culture medium significantly improved the blastocyst rate of parthenogenetic (38.9 +/- 8.8 vs. 27.5 +/- 9.0%) as well as SCNT (22.5 +/- 2.2 vs. 13.8 +/- 3.4%) embryos although cleavage rates were not different (P < 0.05). These blastocysts also had higher hatching ability and contained higher cell number than control blastocysts (P < 0.05). TUNEL labeling revealed that blastocysts cultured in the presence of NEAA were less predisposed to biochemical apoptosis and showed lower indices of TUNEL, fragmentation, and total apoptosis than those cultured in the absence of NEAA (P < 0.05). Real-time qRT-PCR analysis further revealed that NEAA decreased the expression ratio of BAX:BCL-xL and enhanced the relative abundance of IGF2 transcripts. Therefore, our study suggests that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion, hatching, and total cell number and decreasing the apoptosis by positively modulating the expression of embryo survival related genes, similar to those reported for in vivo or in vitro fertilized embryos. Nonessential amino acids improve the yield and quality of cloned and parthenogenetic porcine embryos and modulate the expression of embryo survival related genes.

Comparative study of embryo development and apoptosis vitrification vs. slow freezing of biopsied mouse embryo

Comparative study of embryo development and apoptosis vitrification vs. slow freezing of biopsied mouse embryo

Human ART on chip: improved human blastocyst development and quality with IVF-chip

We developed new IVF Chip system (Mizuno et al, 12th International Academy of Human Reproduction, in Venice). This is the novel report of microfluidic device based “lab on a chip system”, for multiple use (IVF & IVC) for human ART. The goal of our “IVF Chip” are integration of multiple steps of different ART procedure (IVM, IVF and IVC). IRB and Japan society of Obstetrics and Gynecology approved prospective randomized study. In this experiment the frozen/thawed (vitrified) donated human embryos were cultured randomly either IVF Chip or microdrop culture. IVF Chip system: The heart of this device consists in a cell culture room separated in two parts. (Mizuno et al, 12th International Academy of Human Reproduction, in Venice.) Donated Human embryo: Following written informed consent for frozen embryo donation, 2–4 cell human embryos were used for experiment. Donated endometrial cells (EMCs): Frozen EMCs were used for experiment following written informed concent. Culture media: The following media were used: (i) MEMα+ 5% Human Cord Serum (HCS) for EMC preparations, (ii) Global medium (Life Global, USA) + 5% HCS for human embryo IVC. Culture condition: Donated embryos were cultured for 72 h to blastocyst stage in a IVF chip (with EMCs) or 50 μl droplet. They were then cultured at 37°C in an atmosphere of humidified 5% CO2, 5% O2 and 90% N2. Quality evaluation and Total cell numbers of blastocysts: The developed blastocysts quality were evaluated by Gardner's score (criteria) and fixed and stained with 1 μg/ml bisbenzimide, and total nuclei were counted. Development to the blastocyst: 73.7% (14/19) of zygotes developed to at least the blastocyst stage in IVF Chip, the other microdrop control group developed to blastocyst stage slightly lower rate (45.0%, 9/20). Quality evaluation of blastocyst by Gardner's score : 42.9%(6/14) of blastocysts derived from IVF Chip were over 3AB Gardner's score, the other microdrop control group were slightly lower rate (22.2%, 2/9). The total cell counts: Average total cell number of blastocysts derived from IVF Chip group was higher (67.4 ± 17, n = 7) than microdrop groups (55.4 ± 18, n = 5). These results show that this novel IVF Chip embryo co-culture system demonstrate the importance of physical microenvironment on human embryo development.This system may help to improve the efficiencies of human ART.

The effect of osmotic stress on mouse embryos

We hypothesize that long term exposure of culture medium to 37°C in an incubator can cause evaporation and subsequent increase in osmolarity. The objectives of the current study are: - To measure the osmolarity due to evaporation in an incubator. - To evaluate the effect of osmotic stress on mouse embryos by scoring the development and growth rate. - To count the total cell number in blastocysts developed at different osmolarity. Abbreviations: BQ: Blastocyst, ICM: Inner Cell Mass, TE: Trophectoderm, ART: Assisted Reproduction Technology, PI: Propidium iodide

Differential effects of oxygen on hatching of fertilized and parthenogenetic embryos

Embryos exposed to in vitro culture are prone to oxidative damage. Low oxygen tension during early preimplantation development appears beneficial, but is less studied during later development. Moreover, the effects of oxygen on development of parthenogenetic embryos remain unclear. Laboratory studies of fertilized and parthenogenetic mouse embryos, cultured in low (5%) and high (20%, atmospheric) O2, respectively, their rates of development to blastocyst, hatching, and expression profiles of apoptosis, imprinted, oxidation and protease genes. Parthenogenetic embryos (PEs) were achieved by activation of oocytes with strontiuim and cyctochalasin D. Embryos were cultured in KSOM at 37°C under 6% CO2 and 5% or 20% O2. Some embryos were processed for Oct4 expression and apoptosis by TUNEL analysis. Real-time PCR was used for gene expression analysis. Blastocysts developed from zygotes under conditions of low or high O2 were transferred into psuedopregnant recipients for term development. Both PEs and zygotes cultured under 5% or 20% O2 exhibited similar high rates for development to blastocysts (>96%). Apoptosis did not differ between parthenogenetic and fertilized blastocysts, regardless of O2 tension. Notably, blastocysts (n = 77) cultured under 5% O2 showed higher rates of implantation (71% vs. 47%) and live pups (44% vs. 20%), compared to those (n = 89) cultured under 20% O2. Total cell number of PE blastocysts was not affected by O2 tension, but the number of ICM was increased under 5% O2. Zygotes cultured under 5% O2 had markably lower rates of hatching (21%) vs. those under 20% O2 (57%). By contrast, the hatching rate (91%) of PEs cultured under 5% O2 was higher than under 20% O2 (80%). Reduced expression of ERK/MAPK but increased Sod1 was observed for PE blastocysts, with no effects of oxygen tension. High O2 reduced expression of Sod2 in both types of embryos. As expected, expression of the imprinted genes H19 was increased but Snrpn decreased in PE blastocysts, compared to fertilized blastocysts, but neither was affected by oxygen tension. Oxygen tension does not affect development to blastocyst of either fertilized or PEs, but low oxygen tension promotes embryo survival following postimplantation development. Low oxygen inhibits hatching of fertilized blastocysts, but promotes hatching of parthenogenetic blastocysts. The mechanism of oxygen's differential effects on fertilized vs. parthenogenic blastocysts remains to be determined.

Effect of β-mercaptoethanol supplementation on in vitro development of rabbit embryos derived from parthenogenesis and somatic cell nuclear transfer

To investigate effects of β-mercaptoethanol (β-ME) supplementation in in vitro culture (IVC) medium on developmental competence of embryos and total cell numbers (TCN) of blastocysts derived from parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) in rabbits.

Parthenogenetic Activation of Porcine Oocytes and Isolation of Embryonic Stem Cells-like Derived from Parthenogenetic Blastocysts

These experiments were carried out to optimize the parameters of electrical activation, methods of parthenogenetic activation and embryo culture in vitro and meanwhile to isolate embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). These results showed that, as the electric field strength increased from 1.0 to 2.7 kV/cm, the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with that of the 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or 60-μsec pulse duration. The optimum pulse duration was 30-μsec, with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0% vs. 29.9) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0% vs. 59.8%) and blastocysts (19.4% vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than those of G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing 5 μg/ml insulin was significantly higher than that of the control (no insulin) (44.3±9.1 vs. 33.9±11.7). For isolation ofPESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).

Melatonin increases cleavage rate of porcine preimplantation embryos in vitro

Melatonin has been used to promote in vitro embryo development in different species. This study determined the effects of melatonin on in vitro porcine embryo development; in particular, cleavage rate, blastocyst rate, and blastocyst cell number. Starting 5 hr after insemination, porcine zygotes were cultured in porcine zygote medium 3 (PZM-3) culture medium supplemented with melatonin at increasing concentrations (10(-12) M, 10(-9) M, 10(-6) M, 10(-3) M). Melatonin at a concentration of 10(-9) M had a positive effect on cleavage rates, while the highest concentration of melatonin (10(-3) M) significantly decreased cleavage rates. Although blastocyst rates were not increased by 10(-9) M melatonin, blastocyst cell numbers were significantly higher for embryos subjected to 10(-9) M melatonin. The expression levels of the pro-apoptotic gene BAX and anti-apoptotic gene BCL2L1 in blastocysts were not affected by the presence of melatonin in the culture medium. To further study the protective properties of 10(-9) M melatonin against stressful conditions, hydrogen peroxide (0.01 mm) and heat (40 degrees C) were used during embryo culture. The addition of melatonin to embryos subjected to 40 degrees C for 3 hr increased cleavage rates, but had no protective effect for embryos subjected to 0.01 mm H(2)O(2), probably because the physiological levels of melatonin could not counteract the pharmacological levels of H(2)O(2). Our data indicate that 10(-9) M melatonin has a positive effect on porcine embryo cleavage rates and blastocyst total cell numbers and it might have a protective effect against heat stress.

EFFECT OF AMPHIREGULIN ON PREIMPLANTATION DEVELOPMENT OF IN VITRO FERTILIZED AND PARTHENOGENETIC PORCINE EMBRYOS

Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and receptive endometrial epithelium. Although various cellular aspects and molecular pathways of this communication have been identified, one of the proposed molecules for the successful implantation of embryos is amphiregulin (AREG) which is a member of epidermal growth factor (EGF) family and is expressed in the endometrium. The objective of this study was to investigate the effect of AERG supplementation of the in vitro culture medium on the development of porcine in vitro fertilized (IVF) and parthenogenetic embryos by evaluating the rates of cleavage rate, blastocyst formation, and blastocysts cell number. In experiment 1, IVF and parthenogenetic embryos at 1-cell stage were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of AREG (0, 0.5, 5 and 50 ng/ml) for 7 days. There was no difference among all groups in the rates of cleavage and blastocyst formation. Moreover, there were no differences in the total cell number of blastocyst among the IVF and parthenogenetic embryos of 0, 0.5 and 5 ng/ml AREG groups (51.9±19.5 and 51.3±21.1, 61.8±27.8 and 59.3±27.9 and 69.5±27.6 and 56.9±30.3, respectively). However, the total cell numbers of blastocyst in the IVF and parthenogenetic embryos of 50 ng/ml AREG group was significantly higher (p < 0.05) than those of 0 ng/ml AREG groups (78.9±20.3 and 71.2±30.5 vs. 51.9±19.5 and 51.3±21.1, respectively). In experiment 2, the IVF and parhenogenetic embryos at early (1–3 days) or late (4–7 days) stages were cultured in the NCSU-23 medium supplemented with 50 ng/ml AREG and the rate of blastocyst formation and total cell numbers of blastocyst was determined on Day 7. The rates of blastocyst formation did not differ between two groups compared to control group which was cultured in the medium without AREG. However, the total cell numbers of blastocyst at late stage group was significantly higher (p < 0.05) than those of control and in IVF embryos (75.8±27.3 vs. 58.0±23.8, respectively). Besides, the total cell numbers of blastocyst at early stage group and late stage group was significantly higher (p < 0.05) than control group in parthogenetic embryos (77.0±19.0 and 81.2±19.3 vs. 64.4±20.5, respectively). These results suggested that supplementation of AREG in the culture medium, mainly at late preimplantation stage of embryo, improves the subsequent blastocyst formation that exhibit more quality by increasing cell proliferation in the IVF porcine and parthenogenetic embryos. (poster)

Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 μg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 μs) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 μs) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX + Ele); electrical activation followed by exposure to cycloheximide for 6 h (Ele + CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6 h (CHX + Ele + CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for ≥30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX + Ele, Ele + CHX and CHX + Ele + CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX + Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.

In vitro development and cell allocation of porcine blastocysts derived by aggregation of in vitro fertilized embryos

In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to those of their in vivo counterparts. The objective of this study was to increase developmental competence and to gain an understanding of cell allocation in blastocysts derived from the aggregation of four-cell stage porcine embryos produced in vitro. After removal of the zona pellucida, two (2x) and three (3x) four-cell stage embryos were aggregated by co-culturing them in aggregation plates. Five days after aggregation, the developmental ability and the number of cells in the aggregated embryos were determined. The percentage of blastocysts was higher (P < 0.05) in both the 2x and 3x aggregated embryos (66.6% and 72.0%, respectively) compared to that of the 1x embryos and the intact controls (43.1% and 36.4%, respectively). The total cell number of blastocysts also increased in aggregated embryos compared to that of intact controls (2.6-fold for 2x and 3.4-fold for 3x) (P < 0.05). The cells of two differentially stained embryos were started to mix at 72 hr after aggregation. In vitro-fertilized porcine aggregates (2x) were developed to blastocyst with a random distribution of cells from each embryo. The mRNA levels for the oct-4, bcl-xL and connexin 43 genes were higher (P < 0.05) and bak gene were lower (P < 0.05) in both the 2x and 3x aggregated embryos than the intact controls. Therefore, the aggregation of the four-cell stage embryos could be used to improve the quality of porcine preimplantation stage embryos produced in vitro.

Effect of Different Parthenogenetic Activation Methods on the Developmental Competence of in vitro Matured Porcine Oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% ∼ 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 μM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% ∼ 57.3%; 22.3% vs 7.4% ∼ 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% ∼ 46.2%; 18.0% vs 7.1% ∼ 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8–74.4% vs 56.5–57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% ∼ 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3–27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.

Development of bovine–ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine–ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.

37 EFFECTS OF TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Faulty epigenetic reprogramming is a likely major cause of the low success rate observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been reported that treatment of reconstructed mouse embryos with the potent histone deacetylase inhibitor, trichostatin A (TSA), results in significantly increased developmental capacity of SCNT preimplantation embryos and live offspring (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 240, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089; Kishigami et al. 2006 J. Reprod. Dev. 53, 165–170). Studies investigating similar reprogramming capabilities of TSA in bovine SCNT embryos report conflicting results (Akagi et al. 2007 Reprod. Fertil. Dev. 19, 24 abst; Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 48 abst). In this study, the effects of TSA treatment on in vitro development of bovine SCNT embryos were examined. Bovine fetal fibroblasts were cultured under contact inhibition for 2 to 5 days and used as donor cells for SCNT. Oocytes were aspirated from abattoir-derived ovaries, and matured in vitro for 18 h prior to enucleation. Reconstructed SCNT couplets were electrofused, and then activated 24 h post-maturation using 5 µm ionomycin followed by 2 mm dimethylaminopurine (DMAP) for 4 h. SCNT embryos were subjected to 0 (control; C-NT) or 50 nm TSA for 13 h post-ionomycin (hpi) TSAa-NT) or 13 hpi + 6 h starting from 40 hpi (TSAb-NT). IVF embryos were produced as an additional control. All embryos were cultured in KSOM supplemented with 3 mg mL–1 BSA for 7.5 days, with 5% FBS added on Day 3. Experiments were repeated 3 or 7 times, and data were analyzed a -way ANOVA procedure. Developmental rates to the blastocyst stage and total cell number of blastocysts were determined. Total cell numbers were determined by fixing blastocysts in 4% paraformaldehyde, and staining with bisbenzimide 33342, followed by microslide mounting and visualization using an epifluorescence microscope. No difference was observed in cleavage rates among the four treatment groups, C-NT, TSAa-NT, TSAb-NT, and IVF, with the rates being 66%, 75%, 73.1%, and 82.3%, respectively (P = 0.33); nor was any improvement seen in the rate of blastocyst development of TSAa-NT or TSAb-NT over C-NT embryos: 36%, 40.2%, and 30.2%, respectively (P = 0.22). Furthermore, there was no significant difference in mean total cell number of blastocysts among treatment groups: C-NT, 120.2; TSAa-NT, 124.2; TSAb-NT, 129.3; and IVF, 141.1 (P = 0.29). These results suggest that 50 nm TSA treatment immediately following activation does not affect the development of bovine SCNT preimplantation embryos.

Effects of Purple Sweet Potato Anthocyanins on Development and Intracellular Redox Status of Bovine Preimplantation Embryos Exposed to Heat Shock

The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.