scholarly journals 72EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT DEVELOPMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 157
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Luciferase Gene ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Transfected Cells

During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60hpf as detected by their bioluminescence following injection of chicken β-actin/firefly luciferase fusion gene (β-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing β-act/luc+/IRES/EGFP and neor using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20h post maturation. Enucleated oocytes were electrofused with the cells, and activated with calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0hpf, respectively. Cleavage rates were not different among groups (P>0.05). Blastocysts were obtained only from the groups of embryos that were N at 0hpf and P at 60hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.

356 BLASTOCYST DEVELOPMENT FROM DOMESTIC CAT OOCYTES INJECTED WITH DEHYDRATED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 285
Author(s):  
A. Moisan ◽  
S. Leibo ◽  
J. Lynn ◽  
M. Gómez ◽  
C. Pope ◽  
...  
Keyword(s):  
Room Temperature ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Sperm Suspension ◽  
Glass Slides ◽  
Freeze Dried ◽  
Artificial Vagina

Live offspring have been produced by intracytoplasmic sperm injection (ICSI) of dehydrated spermatozoa into mouse and rabbit oocytes (Wakayama and Yanagimachi 1998 Nature Biotechnol. 16, 639; Liu et al. 2004 Biol. Reprod. 70, 1776). The objective of the present study was to determine the capability of dehydrated domestic cat spermatozoa to fertilize oocytes after ICSI. Spermatozoa were collected from three toms by ejaculation into an artificial vagina. Freshly collected samples were layered under 1 mL of Tyrode's salt solution supplemented with HEPES, BSA, glutamine, pyruvate, lactate, and 50 mM ethyleneglyeotetraacetic acid (EGTA) pH = 8.2-8.4, and allowed to swim-up during a 12-min incubation at 38�C. From the upper 600 �L of the sperm suspension, four 100-�L aliquots were collected and placed into 2-mL glass ampoules, plunged into liquid nitrogen (LN2), freeze dried at -43�C to -45�C and 44 to 76 � 10-3 mbar pressure, and stored at 4�C. Four to twelve 10-�L aliquots of the same suspension were placed onto glass slides, air-dried for 30 min, and stored in a desiccator at room temperature. Domestic cat oocytes were collected from minced ovaries and allowed to mature in vitro for 22 to 24 h. After maturation, oocytes were either fertilized in vitro (IVF; n = 36), or sperm injected (ICSI) with fresh or refrigerated (n = 74), freeze-dried (n = 45), or air-dried (n = 45) spermatozoa. After ICSI or IVF, oocytes were cultured in a three-step-sequential medium (G�mez et al. 2004 Cloning Stem Cells 6, 247) for up to 8 days. Cleavage and development to the blastocyst stage was assessed on Days 2 and 8 of culture, respectively. Cleavage rates after IVF (56%), ICSI with freeze-dried (60%), or ICSI with fresh spermatozoa (59%) were higher than those obtained after ICSI with air-dried spermatozoa (35%; P < 0.05). Blastocyst development after ICSI treatments was obtained only with fresh spermatozoa (9%), and was lower than that obtained after IVF (25%; P < 0.05). Recently, one hatching blastocyst was produced when oocytes (n = 18) were exposed to calcium ionophore 2 h after ICSI with freeze-dried sperm. This is the first report that domestic cat embryos can be produced in vitro by injecting oocytes with dried spermatozoa.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P &lt; 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P &lt; 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P &lt; 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P &lt; 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P &lt; 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P &lt; 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

208 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON BLASTOCYST DEVELOPMENT AND POST-TRANSFER SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 183 ◽  
Author(s):  
B. Loureiro ◽  
L. Bonilla ◽  
G. Entrican ◽  
P. J. Hansen
Keyword(s):  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cho Cell ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Cell Supernatant

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been implicated in preimplantation embryo development. Granulocyte-macrophage-CSF improves the proportion of bovine embryos that become blastocysts in vitro (Moraes and Hansen 1997 Biol. Reprod. 57, 1060–1065) and increases blastocyst cell numbers in mice (Robertson et al. 2001 Biol. Reprod. 64, 1206–1215). The long-term goal of the present research was to evaluate the effects of GM-CSF on post-transfer survival of bovine embryos. The experiments used recombinant ovine GM-CSF produced in transfected Chinese hamster ovary (CHO) cells or an equivalent volume of cytokine-free CHO cell supernatant (control). The objective of the first study was to evaluate the effects of GM-CSF on post-transfer survival. Embryos were cultured with 10 ng mL–1 of either GM-CSF or cytokine-free CHO cell supernatant added to culture medium at Day 1 after insemination. Embryos were transferred at Day 7 to lactating dairy cows according to a timed embryo transfer protocol. Pregnancy was evaluated at approximately Day 45 of gestation. There was no significant difference in the proportion of embryos becoming blastocysts at Day 7 after insemination (34.8 v. 37.5% for the control and GM-CSF; SEM = 2.4%). There was also no difference in pregnancy rates between cows receiving control embryos (6/24; 25%) and cows receiving embryos treated with GM-CSF (8/35; 23%). A second study determined the effects of various concentrations of GM-CSF on the development of in vitro-produced embryos to the blastocyst stage. Embryos were cultured in 5% (v/v) oxygen (low oxygen) or atmospheric oxygen (21%, w/v; high oxygen) in the presence of 0, 1, 10, or 100 ng mL–1 of GM-CSF or an equivalent volume of cytokine-free CHO cell supernatant (control). The GM-CSF was added on either Day 1 or Day 5 after insemination. Cleavage rate was accessed on Day 3 after insemination. Stage of development was recorded at Day 7 and Day 8 after insemination. There was no effect of GM-CSF on cleavage rate. Addition of GM-CSF at Day 5 to embryos cultured in low or high oxygen increased the percentage of oocytes that became blastocysts at Day 7 (P < 0.01) and Day 8 (P < 0.01), but addition at Day 1 did not have a significant effect on blastocyst development. The greatest effects of GM-CSF occurred at a concentration of 10 ng mL–1. At this concentration, least squares means for the percentage of oocytes that became blastocysts at Day 7 were 13.9 v. 21.6% (control v. GM-CSF) when GM-CSF was added at Day 5, and 19.5 v. 21.5% when GM-CSF was added at Day 1. The percentage of blastocysts at Day 8 was 20.9 v. 28.7% when GM-CSF was added at Day 5, and 26.7 v. 27.5% when GM-CSF was added at Day 1. In conclusion, GM-CSF can affect the competence of embryos to develop to the blastocyst stage, but at the concentrations and times given, there was no evidence that GM-CSF enhanced embryo survival after transfer.

Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa

2010 ◽  
Vol 22 (2) ◽  
pp. 426 ◽  
Author(s):  
Pablo Bermejo-Álvarez ◽  
Patrick Lonergan ◽  
Detlef Rath ◽  
Alfonso Gutiérrez-Adan ◽  
Dimitrios Rizos
Keyword(s):  
Gene Expression ◽  
Mrna Abundance ◽  
Bovine Embryos ◽  
Same Sex ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Female Embryo ◽  
Male And Female ◽  
Kinetics Of

Using bovine embryos generated in vitro from IVF with X-sorted, Y-sorted and unsorted spermatozoa, we compared the kinetics of male and female embryo development and gene expression between male and female blastocysts. Bovine in vitro-matured oocytes (n = 8858) were fertilised with spermatozoa from each of three different bulls (X-sorted, Y-sorted or unsorted spermatozoa depending on the experiment). The cleavage rate was assessed 24, 27, 30, 33, 36, 40, 44 and 48 h post insemination (h.p.i.) and blastocyst development was recorded on Days 6–9. The relative mRNA abundance of nine genes (GSTM3, DNTM3A, PGRMC1, TP53, BAX, COX2, IGF2R, AKR1B1 and PLAC8) was analysed in male and female Day 7 blastocysts produced with sorted and unsorted spermatozoa from one bull. Cumulative cleavage rate and blastocyst yield were significantly higher in the unsorted group compared with the X- or Y-sorted group from the same bull (P ≤ 0.05). Although differences existed between bulls in terms of cleavage rate, no differences were observed in cleavage rate between X- and Y-sorted spermatozoa within a bull. The blastocyst yield was significantly higher only for Bull 3 when the Y-sorted spermatozoa were used (27.1+2.8 v. 19.1+1.4 for Y- and X-sorted spermatozoa, respectively; P < 0.05). There were no differences in the mRNA abundance of the nine genes analysed between embryos of the same sex produced with sorted or unsorted spermatozoa. However, significant differences in polyA mRNA abundance were observed between male and female blastocysts for three genes (GSTM3, DNMT3A and PGRMC1; P ≤ 0.05). In conclusion, the use of sorted rather than unsorted spermatozoa in IVF significantly delays the onset of first cleavage. Differences were noted between bulls, but not between X- and Y-sorted spermatozoa, and although no differences were found in terms of the mRNA abundance of the nine genes tested between sorted and unsorted spermatozoa, sex-related differences were found in the case of three genes.

scholarly journals 221 TYPICAL HIF1-REGULATED GENES ARE UNALTERED BY OXYGEN IN BOVINE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 261
Author(s):  
A. Harvey ◽  
K. Kind ◽  
J. Thompson
Keyword(s):  
Gene Expression ◽  
Oxygen Concentration ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Expression Of Genes ◽  
Regulated Gene Expression

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) post-compaction oxygen conditions moderate changes in gene expression are observed in the bovine blastocyst (Harvey et al. 2004 Biol. Reprod. 71, in press), compared with 3–4 fold increases in the mouse (Kind et al. 2004 Mol. Reprod. Dev., in press). Specifically, GLUT-1 (Harvey et al. 2004), myotrophin, and anaphase-promoting complex 1 (Harvey et al., unpublished) mRNAs are increased in bovine blastocysts following 2% oxygen culture, compared with those cultured under 20% oxygen. These oxygen-mediated differences in gene expression in the bovine are most likely regulated by hypoxia-inducible factor (HIF)2 transcription factor activity, as we have previously observed that HIF1α protein is not detectable in bovine embryos whereas HIF2α is readily detectable (Harvey et al. 2004). The aim of this study was to determine the effect of post-compaction oxygen concentration on the expression of typically HIF1-regulated and potential HIF2-regulated (suggested from a mouse knockout study; Scortegagna et al. 2003 Nat. Genet. 35, 371) genes in bovine blastocysts. In vitro-produced bovine embryos were generated using standard protocols. Compact morulae were randomly allocated to treatments under 2%, 7%, or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and samples were reverse-transcribed using Superscript II (Invitrogen, Melbourne, Australia). Amplification and analysis of cDNA was achieved by real-time PCR using specific primers and Sybr green PCR master mix (Applied BioSystems, Melbourne, Australia). Statistically significant differences in gene expression were analyzed by ANOVA, P < 0.05. Examination of expression of genes known to be regulated by HIF1 in somatic cells (reviewed by Semenza 2002 Biochem. Pharm. 64, 993) revealed no oxygen-mediated alteration in expression of aldose reductase, cyclooxygenase 2, or inducible nitric oxide synthase. However, the expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001). Expression of glutathione peroxidase, and CuZn- and Mn-superoxide dismutase (putative HIF2-regulated genes) was not influenced by oxygen concentration post-compaction. This study suggests that typical HIF1-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo. These results complement previous observations that HIF1α protein is not detectable in blastocyst-stage bovine embryos, and suggest that LDHA may be an HIF2 target gene in the bovine embryo. As embryo development is influenced by oxygen concentration, levels of LDHA at the blastocyst stage may be used as a marker of oxygen responsiveness.

scholarly journals 62 RELATION OF INTENSITY OF GENE EXPRESSION IN BOVINE RECONSTRUCTED EMBRYOS TO SUBSEQUENT DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 181
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cells ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Critical Event ◽  
Serum Starvation ◽  
Cell Stage ◽  
Sports Science ◽  
Strong Luminescence

During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken β-actin/firefly luciferase fusion gene (β−act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine β−act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with β−act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 μg mL−1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

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

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

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 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s 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.

38 EFFECTS OF TRICHOSTATIN A ON DNA METHYLATION IN CLONED BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 99 ◽  
Author(s):  
D. Iwamoto ◽  
S. Kishigami ◽  
S. Taniguchi ◽  
Y. Abe ◽  
T. Matsui ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Trichostatin A ◽  
Calcium Ionophore ◽  
Serum Starvation ◽  
Image Analyzer ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cloned Bovine

Recently, the efficiency of full-term development of somatic cloned mouse embryos was significantly increased by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have shown that TSA treatment improved the rate of development of the cloned bovine embryos to the blastocyst stage (Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 142 abst). Higher levels of DNA methylation have been shown in early cloned bovine embryos than in in vitro-fertilized (IVF) embryos (Dean et al. 2001 Proc. Nat. Acad. Sci. USA 98, 13734–13738; Santos et al. Curr. Biol. 13, 1116–1121). In this study, we examined the effects of TSA on DNA methylation levels in cloned bovine embryos by immunostaining with an antibody to 5-methyl cytosine (5-MeC). Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days before they were used as donor cells. The cells were electrofused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. Atotal of 131 cloned embryos were produced. The NT embryos were exposed to 0 (control) and 50 nmTSA from the start of activation to 48 h post-activation (hpa). They were then cultured in an mSOF medium. At 60 hpa, only embryos developed to the 8-cell stage were used for assessment of DNA methylation levels. Sixteen TSA-treated, 22 non-treated, and 19 IVF embryos were immunostained with 5-MeC antibody. For quantitative analysis of the DNA methylation levels, 5-MeC signals in the fluorescent images were determined using an image analyzer system (Aqua Cosmos; Hamamatsu Photonics, Shizuoka, Japan). The data were analyzed with Tukey-Kramer post hoc test for multiple comparisons following ANOVA. Relative levels of DNA methylation of TSA-treated cloned and IVF embryos did not differ (P > 0.05), but were lower than those of non-treated cloned embryos (P < 0.05). The results indicate that TSA treatment of cloned bovine embryos leads to a reduction of DNA methylation levels of their genome. The data suggest that the TSA treatment decreased the DNA methylation levels of cloned bovine embryos to the levels of IVF embryos, resulting in improved blastocyst development of the cloned embryos.

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 208
Author(s):  
S. Matoba ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Chi Square

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P > 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P < 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P < 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

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