96 TRICHOSTATIN A IMPROVED THE QUALITY OF RABBIT NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 165 ◽  
Author(s):  
J. Xu ◽  
L.-Y. Sung ◽  
J. Zhang ◽  
X. Tian ◽  
Y. E. Chen ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Trichostatin A ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Developmental Potential ◽  
Number Of Factors ◽  
Nuclear Transplant ◽  
Blastocyst Quality ◽  
And Control

Nuclear reprogramming is dependent upon a number of factors, including chromatin organization and modification. Trychostatin A (TSA), a histone deacetylase inhibitor, was used to increase histone acetylation and to improve reprogrammability in both cattle and mice. The objective of the study was to determine whether TSA could improve the pre-implantational development potential of rabbit nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of FSH and hCG. Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in 10% FBS M199 and confirmed under fluorescence microscopy. Cumulus cells were prepared as nuclear donors for NT; a donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte, and subsequently fused with the oocyte recipient by application of 3 direct current pulses at 3.2 kV cm−1 for a duration of 20 µs/pulse. Fused embryos were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg mL−1 cycloheximide for 1 h. Rabbit NT embryos were cultured in 5 nM TSA-2.5% FBS-B2 medium for 10 h before being transferred into regular medium (FBS-B2). The TSA-treated embryos (5 nM vs. 0 nM) were cultured in 400 µL FBS-B2 medium for 5 days in 5% CO2 in a humidified atmosphere at 38.5°C (initiation of activation = Day 0). NT embryo development to cleaved (2 to 4 cell), morula, and blastocyst stages was evaluated on Day 2, Day 3, and Day 5, respectively. The selected NT blastocysts were counted for cell numbers following Hoechst 33342 epifluorescenin staining. The results (Table 1) showed that there was no difference on pre-implantational development of cloned embryos between TSA-added and control groups (P > 0.05). However, a significantly higher cell number per NT blastocyst was found in the TSA-added group (357 vs. 113; P < 0.05). This indicated that the blastocyst quality in NT embryos was improved with the addition of TSA by increasing histone acetylation activity. The developmental potential of TSA-treated NT embryos to term is under investigation. Table 1.Effects of TSA on the pre-implantational development of cloned rabbit embryos This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.

scholarly journals 66 IN VITRO DEVELOPMENT OF YAK (POEPHAGUS MUTUS) CLONED EMBRYOS BY INTERSPECIES SOMATIC NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 183
Author(s):  
L. Su ◽  
F.L. Du ◽  
L.Y. Sung ◽  
S. Yang ◽  
B.S. Jeong ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Bos Taurus ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Cell Number ◽  
Developmental Potential ◽  
Somatic Nuclear Transfer ◽  
Humidified Air

Interspecies nuclear transfer (NT) is an important tool for preservation of endangered animal species. This study was carried out to clone Yak (Poephagus mutus) embryos by using Yak skin fibroblasts and bovine (Bos taurus) recipient cytoplasts, and to compare the efficiency of YAK interspecies NT (bovine cytoplast-Yak donor cell) and bovine somatic NT (bovine cytoplast-bovine donor cell). Recipient oocytes were extracted from antral follicles of bovine ovaries, and subsequently cultured in maturation medium for 18–20 h in 5% CO2 and 95% humidified air at 39°C. Cumulus cells were removed from the oocytes by vortexing also facilitated further enucleation. Yak skin fibroblast cells were prepared from cultured ear explants of an adult 5-year-old female. Fibroblasts were cultured at passage 6–9 in 10% FBS DMEM at 37°C in 5% CO2 humidified air. The donor cell at a diameter of 19–20 μm was inserted into the perivitelline space of an enucleated oocyte. A bovine female cell line at similar passage number was used for bovine somatic NT as control. Somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV/cm for a duration of 6–10 μs/pulse. Fused embryos were activated in 10 μg/mL cycloheximide and 2.5 μg/mL cytochalasin D in M199 plus 7.5% FBS for 5 h. Reconstructed Yak embryos were cultured in CR1aa plus 6 mg/mL BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2, and 90% N2, and then in 7.5% FBS CR1aa medium for 5 successive days on bovine cumulus monolayers. Expanding and hatching blastocysts on Day 7 were recorded and cryopreserved for further embryo transfer trials. The percentage of cleavage and the development to morulae and blastocysts were statistically analyzed using a General Linear Model (GLM, Univariate, SPSS 9.0, SPSS Inc, Chicago, IL, USA). As indicated in Table 1, the results demonstrated that the efficiencies of fusion rate as well as developmental potential in vitro were significantly higher in the bovine somatic NT group compared to those of the Yak interspecies NT group. However, the morphology and cell number per embryo of interspecies Yak cloned embryos were indistinguishable from those of bovine NT embryos. Our data suggest that bovine oocytes possess the capability of reprogramming/reactivation of the genome from differentiated somatic Yak nuclei. Table 1. Comparison of yak interspecies and bovine somatic nuclear transfer

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

scholarly journals Pyruvate prevents aging of mouse oocytes

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 223-234 ◽  
Author(s):  
Na Liu ◽  
Yan-Guang Wu ◽  
Guo-Cheng Lan ◽  
Hong-Shu Sui ◽  
Li Ge ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Culture Medium ◽  
Accelerated Aging ◽  
Cumulus Cells ◽  
Underlying Mechanism ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Assisted Reproduction Techniques ◽  
Mouse Oocytes ◽  
Oocyte Aging

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus–oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

scholarly journals Inhibition of MEK1/2 and GSK3 (2i system) affects blastocyst quality and early differentiation of porcine parthenotes

PeerJ ◽  
2019 ◽  
Vol 6 ◽  
pp. e5840 ◽  
Author(s):  
Jeongwoo Kwon ◽  
Ying-Hua Li ◽  
Yu-Jin Jo ◽  
YoungJin Oh ◽  
Suk Namgoong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glycogen Synthase ◽  
Epigenetic Modification ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
Cell Number ◽  
Epigenetic Modifications ◽  
Related Gene ◽  
Developmental Potential ◽  
Blastocyst Quality

Inhibition of both MEK1/2 and glycogen synthase kinase-3 (GSK3; 2i system) facilitates the maintenance of naïve stemness for embryonic stem cells in various mammalian species. However, the effect of the inhibition of the 2i system on porcine early embryogenesis is unknown. We investigated the effect of the 2i system on early embryo development, expression of pluripotency-related genes, and epigenetic modifications. Inhibition of MEK1/2 (by PD0325901) and/or GSK3 (by CHIR99021) did not alter the developmental potential of porcine parthenogenetic embryos, but improved blastocyst quality, as judged by the blastocyst cell number, diameter, and reduction in the number of apoptotic cells. The expression levels of octamer-binding transcription factor 4 and SOX2, the primary transcription factors that maintain embryonic pluripotency, were significantly increased by 2i treatments. Epigenetic modification-related gene expression was altered upon 2i treatment. The collective results indicate that the 2i system in porcine embryos improved embryo developmental potential and blastocyst quality by regulating epigenetic modifications and pluripotency-related gene expression.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 199
Author(s):  
L.-Y. Sung ◽  
C.-H. Chen ◽  
T.-A. Lin ◽  
L.-J. Sung ◽  
H.-Y. Su ◽  
...  
Keyword(s):  
Polar Body ◽  
Fluorescent Microscopy ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Adult Rabbit ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
First Polar Body

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

scholarly journals Antioxidants increase blastocyst cryosurvival and viability post-vitrification

2020 ◽  
Vol 35 (1) ◽  
pp. 12-23 ◽  
Author(s):  
Thi T Truong ◽  
David K Gardner
Keyword(s):  
Oxidative Stress ◽  
Histone Acetylation ◽  
Lipoic Acid ◽  
Fetal Development ◽  
Limb Development ◽  
Cell Mass ◽  
Cell Number ◽  
Crown Rump Length ◽  
Developmental Potential

Abstract STUDY QUESTION What is the effect of antioxidants acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid (A3) in vitrification and warming solutions on mouse blastocyst development and viability? SUMMARY ANSWER The combination of three antioxidants in vitrification solutions resulted in mouse blastocysts with higher developmental potential in vitro and increased viability as assessed by both an outgrowth model in vitro and fetal development following uterine transfer. WHAT IS KNOWN ALREADY The antioxidant combination of acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid present in IVF handling and embryo culture media has significant beneficial effects on mouse embryo and fetal development, especially under oxidative stress. STUDY DESIGN, SIZE, DURATION The study was a laboratory-based analysis of an animal model. Rapid cooling through vitrification was conducted on F1 mouse blastocysts, with antioxidants (A3) supplemented in vitrification and/or warming solutions, followed by culture and embryo transfer. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Pronucleate oocytes were collected and cultured in groups to Day 4 blastocysts. Expanded blastocysts were vitrified and warmed in solutions with and without the A3 antioxidants and cultured for a further 24 h. Blastocyst cell number and allocation, apoptosis and histone acetylation levels were all quantified, and viability through outgrowths and transfers assessed. MAIN RESULTS AND THE ROLE OF CHANCE Mouse blastocysts vitrified with no antioxidants had significantly lower cell numbers (P  &lt; 0.001) and higher apoptotic cells (P  &lt; 0.05) compared to non-vitrified embryos. Addition of combined A3 antioxidants to the vitrification and warming solutions resulted in a significant increase in inner cell mass cell (ICM) number (P  &lt; 0.001) and total cell number (P  &lt; 0.01), and an increase in outgrowth area (P &lt; 0.05), which correlated with the increased fetal weight (P &lt; 0.05), crown rump length (P &lt; 0.05) and limb development (P &lt; 0.05) determined following transfer compared to embryos with no antioxidants. Furthermore, while blastocyst vitrification significantly reduced acetylation levels (P &lt; 0.05) compared to non-vitrified embryos, the inclusion of A3 antioxidants helped to ameliorate this. LIMITATIONS, REASONS FOR CAUTION Embryo development was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS Results in this study demonstrate that vitrification and warming of blastocysts have significant detrimental effects on embryo histone acetylation and subsequent viability. The presence of antioxidants in the vitrification solutions helps to alleviate the negative effects of cryopreservation. Our data indicate that antioxidants need to be present in the medium at the time of exposure to increased oxidative stress associated with vitrification and that prior exposure (i.e. during culture or IVF alone) is insufficient to protect cells against cryo-induced injury. Hence, A3 antioxidants may assist in maintaining the viability of vitrified human embryos in ART through the reduction of oxidative stress. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Enhancement of histone acetylation by trichostatin A during in vitro fertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 209-215 ◽  
Author(s):  
Shuntaro Ikeda ◽  
Atsuhiro Tatemizo ◽  
Daisaku Iwamoto ◽  
Shunji Taniguchi ◽  
Yoichiro Hoshino ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Bovine Oocytes

SummaryHistone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

scholarly journals Developmental ability of cloned embryos from neural stem cells

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 849-857 ◽  
Author(s):  
Eiji Mizutani ◽  
Hiroshi Ohta ◽  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Takafusa Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Success Rate ◽  
Sertoli Cells ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cloning Efficiency ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Undifferentiated State

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2–3.5%). Although thein vitrodevelopmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula–blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point forin vitrodevelopment of cloned embryos depends on the donor cell type.

298 EFFECTS OF TRICHOSTATIN A DURING IN VITRO FERTILIZATION OF BOVINE OOCYTES ON SUBSEQUENT DEVELOPMENT, CELL NUMBER, AND ALLOCATION OF RESULTING EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 164
Author(s):  
S. Ikeda ◽  
K. Saeki ◽  
A. Tatemizo ◽  
D. Iwamoto ◽  
A. Kasamatsu ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Histone Acetylation ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Cell Mass ◽  
Cell Number ◽  
Vitro Fertilization ◽  
Cell Numbers ◽  
Bovine Oocytes

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization or of transferred cell genomes after nuclear transfer to establish a totipotent state for normal development. In the fertilization of bovine oocytes, asynchronous histone acetylation occurs during pronuclear formation in the manner that modification of the paternal genome precedes that of the maternal genome (Wee et al. 2006 J. Biol. Chem. 281, 6048–6057). In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse ovaries were in vitro-matured (IVM) for 21 h in TCM-199 supplemented with 5% v/v FCS, 0.5 mM sodium pyruvate, 0.02 AU mL-1 FSH, and 1 �g mL-1 estradiol-17β at 39�C under 5% CO2 in air. After IVM, the oocytes were subjected to IVF with 3 � 106 mL-1 of Percoll gradient-selected sperm in a defined medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were freed from cumulus cells and cultured in mSOF medium until 168 h post-insemination (hpi) at 39�C under 5% CO2, 5% O2, and 90% N2 with high humidity. Cleavage and blastocyst development were assessed at 48 and 168 hpi, respectively. Inner cell mass (ICM) and trophectoderm (TE) of blastocysts were differentially stained by the method of Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29) to assess cell number and ICM/TE ratio. Experiments were replicated 3 times. Data are presented as means � SEM and statistically analyzed by multiple comparison with the Holm method. Rates of cleavage (0 nM: 71.0 � 7%, n = 102; 5 nM: 75.5 � 5%, n = 106; 50 nM: 68.8 � 6%, n = 105; and 500 nM: 71.7 � 4%, n = 98) and blastocyst formation (21.4 � 5%, 22.3 � 6%, 17.8 � 2%, and 18.2 � 2%, respectively) were similar among the groups. However, 500 nM TSA significantly (P &lt; 0.05) increased ICM and total cell numbers (59.8 � 4 and 143.5 � 7, respectively, n = 31) compared with the control (43.1 � 3 and 120.9 � 7, n = 31). In addition, ICM/TE ratios were higher in the 50 nM (0.81 � 0.08, n = 29) and 500 nM (0.92 � 0.2, n = 31) groups than in the control (0.59 � 0.04, P &lt; 0.05). These results suggest that TSA treatment during IVF of bovine oocytes does not affect the blastocyst rate but alters the cell numbers and their allocation to ICM and TE. Overriding epigenetic modification of the genome during fertilization may have a carryover effect on cell proliferation and differentiation in pre-implantation embryos. This study was supported by a grant from Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.

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