Blastocyst Total Cell Number Research Articles

28 TREATMENT WITH SUBEROYLANILIDE HYDOXAMIC ACID OR SODIUM BUTYRATE ON PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DERIVED FROM KIDNEY CELLS OF HUMAN HEME OXYGENASE-1 TRANSGENIC PIG

Because somatic cell nuclear transfer (SCNT) is influenced by many factors concerning a series of various steps, the cloning efficiency is low in so many species and it seems to be more serious in production of transgenic (TG) animals. Reprogramming of donor nucleus is one of the important factors that affects the developmental competence of SCNT embryos, and several epigenetic remodelling drugs have been used to improve the cloning efficiency. In this study, we examined the effect of suberoylanilide hydoxamic acid (SAHA) or sodium butyrate (NaBu) treatment on the development of porcine SCNT embryos derived from kidney cells of TG pig. Fully confluent porcine kidney cells expressing the human heme oxigenase-1 gene were used for nuclear donor. For SCNT, matured oocytes with 1st polar body were enucleated, electrically fused, and activated 1 h after fusion (Song et al. 2009 Mol. Reprod. Dev. 76, 611–619). Then, SCNT embryos were incubated in postactivation medium [PA; porcine zygote medium-5 (PZM-5) supplemented with 0.5% dimethyl sulfoxide] for 4 h (control), PA with 0.4 μg mL–1 demecolcine for 4 h (Dc), PA with 0.5 μM SAHA for 9 h (SAHA), or PA with 1 mM NaBu for 9 h (NaBu). After postactivation treatment, SCNT embryos were cultured in fresh PZM-5 for 7 days. The embryos were examined for cleavage and blastocyst formation on Days 2 and 7, respectively (the day of SCNT was designated Day 0). Total cell number of blastocysts was examined by counting the number of nuclei stained with Hoechst 33342 under ultraviolet light. Complementary DNA synthesised with total RNA extracted from blastocysts were used for qRT-PCR to determine HDAC2, HDAC6, and GAPDH gene expression. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism version 5.01 (Graphpad Software, San Diego, CA, USA). The cleavage rates (77.0–82.9%) of treated embryos were not different from that of control embryos (79.0%). Blastocyst formation was slightly increased in Dc- (36/132, 27.3%), SAHA- (34/125, 28.6%), and NaBu- (36/133, 27.3%) treated embryos than in control embryos (32/128; 25.0%), but the difference was not significant. Total cell numbers (45.2–47.5) of treated embryos were not different from that of control embryos (51.8). Expression of HDAC2 was higher in SAHA-treated embryos than in control and Dc-treated embryos (P < 0.05), but it was not different from that of NaBu-treated embryos. The relative expression of HDAC6 transcript was increased in SAHA- and NaBu-treated embryos, but there was no significant difference among all groups. Although SAHA or NaBu did not improve the pre-implantational development of porcine SCNT embryos derived from kidney cells of TG pig as assessed in this study, additional studies are needed to determine the effect of SAHA or NaBu on gene expression of pig TG embryos and developmental competency following embryo transfer according to the origin of donor cells. This study was supported by IPET (#311011-05-2-SB010), MOTIE (#10033839-2012-21) and the TS Corporation.

133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

24 IMPROVEMENT OF PORCINE CLONED EMBRYONIC STEM-LIKE CELLS DERIVATION BY ZONA-MEDIATED EMBRYO AGGREGATION

Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142±21.8 vs. 117±29.7 and 120±25.2; P<0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P<0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P<0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO2in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.

Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows on oocyte developmental competence

Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin of Escherichia coli origin (G-), or Gram-positive toxin of Staphylococcus aureus origin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G- and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium for in vitro embryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G- administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G- groups than in controls (P<0.05). Blastocyst formation rate (7-8 days postfertilization) was lower in the G- group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G- group, relative to controls. Examining mRNA relative abundance in oocytes and early embryos revealed mastitis-induced alterations in PTGS2 (COX2), POU5F1, and HSF1 but not in SLC2A1 (GLUT1) or GDF9. Results indicate a differential disruptive effect of mastitis induced by G- and G+ on oocyte developmental competence in association with alterations in maternal gene expression.

Effect ofin vitroManipulation on Development of Pre-implantation Embryos: Lecture of Light Effects

Under normal milieu, mammalian oocytes are fertilized, and the zygotes develop into blastocysts in the female reproductive tract. After pick-up of oocytes, exposure to light during in vitro manipulation is unavoidable. Development of mouse zygotes to blastocysts is unaffected by exposure to cool white fluorescent light for 15 min, but the incidence of apoptotic cells in blastocysts is significantly increased by the light exposure. The blastocysts that develop from zygotes exposed to light develop to term fetuses at a lower rate than those developed from zygotes shielded from light. The incidence of apoptoic cells in blastocysts is significantly decreased by addition of SB20358, p38MAPK inhibitor, or Q-VD-Oph, caspase inhibitor. Furthermore, when HB-EGF is added to the culture medium, the total cell number of blastocysts developing from zygotes exposed to light increases, and the incidence of apoptotic cells in blastocysts decreases. These blastocysts develop to term fetuses at the same rate as those developed from zygotes shielded from light. In vitro conditions are stressful, and are not the best environment for eggs. Light is one of the physical factors affecting the function of mammalian embryos and extreme care should be taken concerning its effects.

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores.

Low Oxygen Tension and Relative Defined Culture Medium with 3, 4‐Dihydroxyflavone are Beneficial for Yak–Bovine Interspecies Somatic Cell Nuclear Transfer Embryo

With an aim to improve the efficiency of yak-bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak-bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P<0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (P<0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress-related genes (GPX-1, SOD-1, CAT and GSS) (P<0.05). 3, 4-Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (P<0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak-bovine iSCNT embryo during IVC.

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro-cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT.

Effects of Trichostatin A on In Vitro Development of Porcine Parthenogenetic and Nuclear Transfer Embryos

Developmental potential of cloned embryos is related closely to epigenetic modification of somatic cell genome. The present study was to investigate the effects of applying histone deacetylation inhibitor, trichostatin A (TSA) to activated porcine embryos on subsequent development of porcine parthenogenetic and nuclear transfer embryos. Electrically activated oocytes were treated with 5 nM TSA for different exposure times (0, 1, 2 and 4 hr) and then the activated embryos were cultured for 7 days. The reconstructed embryos were treated with different concentrations of 0, 5, 10 and 25 nM TSA for 1 hr. Also 5 nM TSA was tested with different exposure times of 0, 0.5, 1, 2 and 4 hr. And fetal fibroblast cells were treated with 50 nM TSA for 1, 2 or 4 hr and with 5 nM TSA for 1 hr. Cumulus-free oocytes were enucleated and reconstructed by TSA-treated donor cells and electrically fused and cultured for 6 days. In parthenogenetic activation experiments, 5 nM TSA treatment for 1 hr significantly improved the percentage of blastocyst developmental rates than the other groups. Total cell number of blastocysts in 1 hr group was significantly higher than other groups or control. Similarly, blastocyst developmental rates of porcine NT embryos following 5 nM TSA treatment for 1 hr were highest. And the reconstructed embryos from donor cells treated by 50 nM TSA for 1 hr improved the percentage of blastocyst developmental rates than the control group. In conclusion, TSA treatment could improve the subsequent blastocyst development of porcine parthenogenetic and nuclear transfer embryos.

Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro

In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium (mCZB) as control group. The experimental group (Ica group) was supplemented with 0.6 μg mL−1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21: pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group ((85.14±6.57)% vs. (72.04±11.58)%; (82.50±7.11)% vs. (66.80±11.70)%, respectively, P<0.01). The total cell number of blastocysts had extreme difference between Ica group and control group ((61.40±9.64) vs. (46.23±4.50), P<0.01). The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group ((1.47±0.51) vs. (2.94±0.66); (2.40±0.27)% vs. (6.25±0.62)%, respectively, P<0.01). Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 (P<0.01), down-regulated pro-apoptotic Caspase3 (P<0.05) and PTEN (P<0.01), up-regulated anti-apoptotic Bcl-2 (P<0.01). In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro-apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.

Replacement of glutamine with the dipeptide derivative alanyl-glutamine enhances in vitro maturation of porcine oocytes and development of embryos

The presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.

Effect of Glycosaminoglycans on In vitro Fertilizing Ability and In vitro Developmental Potential of Bovine Embryos.

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 μg/ml of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

Insulin-like growth factor 1 acts as an autocrine factor to improve early embryogenesis in vitro

Consideration of embryo-derived growth factors, such as IGF1, is important when culturing an embryo in an in vitro fertilization (IVF) setting, or when studying the effect of growth factors on embryo development in vitro. Addition of IGF1 to the culture medium has been reported to cause a range of developmental responses in preimplantation mouse embryos. This variability may be due to culture of embryos in suboptimal culture media and at different culture densities/volumes. This study examined the role of exogenous and autocrine IGF1 on mouse preimplantation development in vitro, by treatment of embryos with an IGF1R neutralising antibody (IGF1R nAb) under low density (1 embryo/100microl) or high density (1 embryo/1microl) culture conditions. At low density, IGF1R nAb reduced development to the blastocyst stage, hatching, and total cell numbers in blastocysts and increased the number of apoptotic cells in blastocysts, suggesting that autocrine IGF1 signalling is occurring, even at low density. This signalling is independent of IGF1 present in the zona pellucida, since culturing embryos in the absence of their zona pellucida had no effect on blastocyst development. Addition of 10 ng/ml IGF1 increased blastocyst development at low density, but decreased hatching at high density. Similarly, high levels of exogenous IGF1 at low density decreased hatching. IGF1 appears to play a role in cell survival and treatment of blastocysts with IGF1 increased Akt phosphorylation. The IGF1R antagonist picropodophyllin was also used in this study, but was found to have non-specific effects on the mitotic spindle. In conclusion, IGF1 is an important growth factor for the improvement of preimplantation development; however, for optimal development the total amount of IGF1 present must be tightly controlled.

Effects of ghrelin on developmental competence and gene expression of in vitro fertilized ovine embryos

The objective was to determine the effects of various ghrelin concentrations (0, 10, 50, and 250 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on ovine embryo development and expression patterns of genes involved in transcription regulation (OCT4), glucose transport (GLUT1), pregnancy recognition (IFNT), and ghrelin receptor (GHSR-1a). Rates of cleavage and blastocyst formation were decreased when oocytes were matured with 250 ng/mL ghrelin compared with 0, 10, or 50 ng/mL ghrelin (P < 0.05). Addition of 50 ng/mL ghrelin during IVC or during IVM and IVC significantly increased blastocyst rates (35.3% and 36.7% vs. 26.9% and 26.8%, respectively) and total cell numbers per blastocyst (110.4 and 108.3 vs. 81.2 and 77.4) compared with 0 and 10 ng/mL ghrelin. However, a high concentration (250 ng/mL) of ghrelin during IVM and IVC decreased cleavage, blastocyst rate, and total cell number of blastocyst compared with low concentrations (P < 0.05). Relative abundances of GLUT1 and IFNT transcripts were higher in blastocysts treated with 50 ng/mL ghrelin during IVC compared with other concentrations (P < 0.05). Expression of GHSR-1a was higher when 10 ng/mL ghrelin was added during IVM (0.079) or during IVM and IVC (0.053) compared with other treatments. However, addition of ghrelin at higher concentrations (50 or 250 ng/mL) reduced relative abundances of GHSR-1a transcripts (0.032 and 0.039, P < 0.05). In conclusion, appropriate concentrations of ghrelin promoted ovine blastocyst formation in vitro and increased expression of GLUT1, IFNT, and GHSR-1a genes in blastocysts, although a high concentration of ghrelin suppressed embryo development.

Effect of Trans-ε-Viniferin on &lt;i&gt;In Vitro&lt;/i&gt; Porcine Oocyte Maturation and Subsequent Developmental Competence in Preimplantation Embryos

ABSTRACTTrans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoid family that has been isolated from Vitis amurensis, one of the most common wild grapes in Asia. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after in vitro fertilization (IVF) or parthenogenesis (PA). We observed that trans-ε-viniferin treatment during IVM did not improve nuclear maturation rates of oocytes in any group, but significantly increased (P<0.05) intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS) levels in the 0.5 µM treatment group. Trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (P<0.05) expression of DNA methyltransferase-1 (DNMT1) mRNA in the 0.5 µM treatment group as compared with the control group. However, the expression of essential transcriptional and apoptosis-related genes did not significantly differ from that of the control. In cumulus cells, pro-apoptosis gene expressions were changed as apoptosis decreased. Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after PA, but total cell numbers were significantly higher (P<0.05) in the 0.5 and 5.0 µM treatment groups compared with those in the control group. IVF embryos showed similar results. In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased the total cell number of blastocysts, possibly by increasing intracellular GSH synthesis, reducing ROS levels, increasing DNMT1 gene expression of oocytes and decreasing pro-apoptosis gene expressions of cumulus cells.

37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P &lt; 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P &lt; 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

40 PIG CLONING AND GENE EXPRESSION PATTERNS IN MINIPIG ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are very interesting; they are easy to harvest and can expand to generate millions of cells from a small quantity of fat. The ASC are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). In this study, we investigated the expression patterns of several genes (Oct-4, Nanog, Sox2, Dnmt1, and Dnmt3b) in minipig ASC, and whether ASC can be a suitable donor cell type for producing cloned pigs. For the study, we respectively isolated ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) from a 6-year-old female minipig. The ASC were attached to a plastic dish with a fibroblast-like morphology, expressed cell-surface marker characteristics of stem cells, and underwent osteogenic, adipogenic, and neurogenic differentiation when exposed to specific differentiation-inducing conditions. To observe gene expression, total RNA was extracted from ASC, FF, and ASF, respectively, and was used for reverse transcription (RT). After RT, real-time PCR was performed to investigate the expression of Oct-4, Sox2, Nanog, Dnmt1, and Dnmt3b. The expression of β-actin was measured and used as an endogenous control. In the following experiment, we carried out SCNT using ASC, ASF, and FF. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. Statistical analysis was performed using one-way ANOVA (GraphPad Prism version 5). As a result, the relative abundance of DNMT1 in ASC (1.9 ± 0.9) was significantly higher than that in FF and ASF (0.1 ± 0.2 and 1.0 ± 0.5, respectively; P &lt; 0.05), but no significant difference in expression of the DNMT3b gene was observed. Interestingly, the quantity of Oct-4 was significantly higher in FF and ASC than in ASF (2.8 ± 0.4 and 2.9 ± 0.5 v. 1.0 ± 0.1, respectively; P &lt; 0.05), and Sox2 showed significantly higher expression in ASC (3.7 ± 0.5) than in ASF and FF (1.0 ± 0.1 and 1.4 ± 0.6, respectively; P &lt; 0.05). Nanog expression was similar in ASF, FF, and ASC. After SCNT, the developmental competence to blastocysts did not differ among the 3 groups (ASF: 7.0 ± 0.2%, FF: 16.15 ± 6.1%, and ASC: 11.1 ± 0.7%). However, total cell numbers of blastocysts derived from ASC and FF were significantly higher in ASF (89.0 ± 7.9 and 105.0 ± 5.5 v. 57.5 ± 5.2, respectively). In conclusion, the present study revealed that minipig ASC and minipig FF possess slightly different gene expression patterns and ASC have potential in terms of in vitro development and blastocyst formation ability similar to ASF and FF. This study was supported by IPET (no. 311011-05-1-SB010), RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), Institute for Veterinary Science, and the BK21 program.