Buffalo (Bubalus bubalis) SCNT embryos produced from somatic cells isolated from frozen–thawed semen: effect of trichostatin A on the in vitro and in vivo developmental potential, quality and epigenetic status

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 549-553 ◽  
Author(s):  
Naresh L. Selokar ◽  
Monika Saini ◽  
Himanshu Agrawal ◽  
Prabhat Palta ◽  
Manmohan S. Chauhan ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Somatic Cells ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Global Level ◽  
Third Trimester ◽  
Developmental Potential

SummaryThis study examined the effects of trichostatin A (TSA) treatment of reconstructed buffalo embryos, produced by hand-made cloning using somatic cells isolated from over a decade old frozen–thawed semen, on their in vitro and in vivo developmental competence, quality and epigenetic status. Following treatment of reconstructed embryos with TSA (0, 50 or 75 nM) for 10 h prior to culture, the cleavage (100.0 ± 0, 94.5 ± 2.3 and 96.1 ± 1.2%, respectively) and blastocyst rate (50.6 ± 2.3, 48.4 ± 2.7 and 48.1 ± 2.6%, respectively), total cell number (275 ± 17.4, 289 ± 30.1 and 317 ± 24.2, respectively) and apoptotic index (5.6 ± 0.7, 3.4 ± 0.9 and 4.5 ± 1.4, respectively) were not significantly different among the three groups. However, TSA treatment increased (P < 0.05) the global level of H4K5ac and decreased (P < 0.05) that of H3K27me3 in blastocysts whereas the global level of H3K18ac was not affected significantly. Transfer of embryos treated with 75 nM TSA (n = 10) to recipients resulted in two pregnancies (20%), one out of which was aborted in the second and the other in the third trimester whereas transfer of control embryos (n = 20) or those treated with 50 nM TSA (n = 12) did not result in any pregnancy. In conclusion, these results suggest that TSA treatment of cloned buffalo embryos produced using somatic cells isolated from frozen–thawed semen improved their epigenetic status but not the in vitro developmental potential and offspring rate.

The developmental potential of mouse somatic cell nuclear-transferred oocytes treated with trichostatin A and 5-aza-2′-deoxycytidine

Zygote ◽  
2009 ◽  
Vol 17 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Yuta Tsuji ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Treatment Duration ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

SummaryTo facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.

scholarly journals Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

2020 ◽  
Author(s):  
Vera A van der Weijden ◽  
Meret Schmidhauser ◽  
Mayuko Kurome ◽  
Johannes Knubben ◽  
Veronika L Flöter ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Early Stage ◽  
Developmental Competence ◽  
Molecular Signaling ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Molecular Signaling Pathways ◽  
Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

scholarly journals In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development

2017 ◽  
Vol 29 (9) ◽  
pp. 1667 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Gene Markers ◽  
Developmental Potential ◽  
Junction Protein ◽  
Mitochondrial Distribution ◽  
Potential Biomarkers

In vivo-matured cumulus–oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

65 EFFECT OF TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENT OF DONOR CELLS, FUSED EMBRYOS, OR BOTH ON THE DEVELOPMENTAL COMPETENCE, QUALITY, AND EPIGENETIC STATUS OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 162
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Species Conservation ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Epigenetic Modifiers ◽  
Donor Cells ◽  
Significant Difference ◽  
Transgenic Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology in buffalo for multiplication of elite animals, species conservation, and production of transgenic embryos for therapeutic applications. However, the cloning efficiency obtained in this species is very low, which might be due to improper reprogramming of donor cells after SCNT. Treatment of donor cells or fused embryos or both with epigenetic modifiers might be a suitable approach to improve the ability of donor cells to be reprogrammed. The present study was aimed at examining the effects of treatment of donor cells (24 h before SCNT) or fused embryos (10 h post-electrofusion) or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence, quality, and epigenetic status of buffalo embryos produced by hand-made cloning (HMC) as described earlier (Saini et al. 2014 Reprod. Fertil. Dev. doi: 10.1071/RD14176). The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher’s least significant difference test. The blastocyst rate was significantly higher (P < 0.05) and the apoptotic index was significantly lower (P < 0.05) in embryos produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls (Table 1). However, the cleavage rate and the total cell number were not significantly different among all the groups. The global level of H3K18ac, examined by immunofluorescence staining, was higher (P < 0.05) and that of H3K27me3 was lower (P < 0.01) in blastocysts produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls. These results show that treatment of donor cells, fused embryos, or both with TSA + 5-aza-dC improves the developmental competence and quality, and alters the epigenetic status of buffalo embryos produced by HMC. However, the effects of treatment with these epigenetic modifiers on the pregnancy rate require further studies. Table 1.Effect of treatment of donor cells, fused embryos, or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence and level of apoptosis in cloned embryos

scholarly journals 127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

2004 ◽  
Vol 16 (2) ◽  
pp. 186
Author(s):  
J.O. Gjørret ◽  
P. Maddox-Hyttel
Keyword(s):  
Production Systems ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Embryo Cell ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&amp;T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&amp;T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&amp;T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to&lt;0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P&lt;0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

157 AGGREGATION AND DEVELOPMENT OF RABBIT CLONED EMBRYOS WITH ZYGOTIC, TETRAPLOID, AND PARTHENOGENETIC EMBRYO IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 237
Author(s):  
C.-H. Chen ◽  
T.-A. Lin ◽  
H.-Y. Su ◽  
Y.-S. Sung ◽  
L.-J. Sung ◽  
...  
Keyword(s):  
Standard Procedure ◽  
Cell Number ◽  
Cell Counting ◽  
Culture In Vitro ◽  
Developmental Potential ◽  
Counting Data ◽  
Parthenogenetic Embryo ◽  
Different Origin

The rabbit compared to other domestic animals, such as cattle and sheep, was a relatively more difficult species to clone. One of the major reasons may be attributed to the low cell number in cloned embryo before implantation. This study was designed to aggregate 2 nuclear transfer (NT) embryos and one with a different origin, determine their developmental potential in vitro, and finally examine the cell number of aggregated embryos. NT was performed with our standard procedure using in vivo derived oocytes and donor cells from adult skin fibroblasts. Zygotes (ZY) were collected from does at 18 h post-hCG and mating. Parthenogenetic (PA) embryos were generated from oocytes with activation protocol, whereas tetraploid embryos (4N) were prepared by fusing fertilized embryos at 2-celled stage into 1-celled stage by electrical pulse. All of the embryos were cultured for 20-24 h into 4-/8-celled stage prior to aggregation. Zona pellucida was then removed and 2 NT embryos were aggregated with 1 embryo originated from ZY, PA, and 4N groups in a depressed droplet containing culture medium. Aggregated embryos were cultured for another 48 h (total 3 days, initiation of activation = Day 0) before being fixed for cell counting. Both single embryo from NT, ZY, PA, and 4N and 3 embryo aggregates (3X) from the same category were used as controls for NT aggregation. The results of 3-day embryo culture in vitro showed that the development of aggregated embryos to blastocyst (BL) stage was 2 NT + ZY, 68.6% (n = 35); 2 NT + 4N, 91.7% (n = 36); and 2 NT+PA, 37.5% (n = 24), whereas 3X aggregates developed to BL at a rate of ZY, 100% (n = 24); 4N, 100% (n = 19); and PA, 100% (n = 14). The BL rates of single embryo control developed into early BL were ZY, 100% (n = 34) and 4N, 93% (n = 36); however, NT and PA developed slower, only 45.4% NT (n = 187) and 79.2% PA (n = 72) to compacted morula/early BL stage. Cell counting data (Table 1) showed that there was no difference in cell number per embryo between NT and PA, whereas ZY and 4N possessed significantly higher cell number than NT and PA (128-162 v. 52-61, P < 0.05) for single embryo category. In the 3X aggregation group, significantly higher cell number per aggregated embryo was found in ZY and 4N compared to that in PA (549-564 v. 196, P < 0.05). More importantly, there was significantly higher cell number found in 2 NT + 4N embryo than that in single 4N (293 v. 162, P < 0.05). This result demonstrated that 2 cloned embryos had propagated and incorporated into 1 tetraploid embryo during pre-implantational development. The next step is to study how NT embryos successfully interact within the aggregated embryo during further development and differentiation, in order to increase the birth rate of clones. Table 1.The cell number of aggragated rabbit embryos between NT and other embryos of different origin after 3 days of culture in vitro Supported by NIH 5R44HL091605-03.

In vitro culture and mtDNA fate of ibex–rabbit nuclear transfer embryos

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 233-240 ◽  
Author(s):  
Yan Jiang ◽  
Tao Chen ◽  
Chang-Long Nan ◽  
Ying-Chun Ouyang ◽  
Qing-Yuan Sun ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Developmental Stages ◽  
Somatic Cells ◽  
Developmental Competence ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Capra Ibex ◽  
Mt Dna ◽  
Developmental Potential

Rabbit oocyte can be used as the recipient in interspecies somatic cell nuclear transfer (iSCNT). This work was undertaken in order to study the developmental competence of Capra ibex somatic cells reprogrammed by rabbit oocytes and the fate of mitochondria in iSCNT embryos. Metaphase II (MII) oocytes from superovulated rabbit were used as nuclear recipients. The nuclear donors were Capra ibex somatic cells with different proliferative status: population doubling time (PDL)=15±2 (group 1), 35±2 (group 2), 55±2 (group 3) and 70±2 (group 4). Oocytes reconstructed with electrical pulses (2.1 kV/cm, 10 μs, 2 times) were activated (1.4 kV, 20 μs, 2 times) and then cultured in Medium199 containing 10% fetal bovine serum at 38.5 °C, 5% CO2 in air. In groups 1, 2, 3 and 4, the fusion rates were 35.83%, 66.03%, 65.40% and 35.35%, respectively. Similar cleavage rates were observed among the four groups. However, the developmental potential to morula/blastocyst from early nuclear donor embryos (16.42%/10.45%) was significantly higher (p < 0.05) than in terminal donor embryos (9.52%/3.81%). Polymerase chain reaction analysis of the mitochondrial (mt) DNA cytb gene demonstrated that mtDNAs from ibex and rabbit could be detected at various developmental stages before implantation. In conclusion, our results provide some original information about rescuing Capra ibex using the iSCNT technique. These results indicate that: (1) enucleated rabbit oocytes make Capra ibex fibroblast nuclei reprogramme; (2) the proliferative status of donor cells affects the efficiency of iSCNT; and (3) rabbit ooplasm rescues the donor-derived mtDNAs, resulting in mtDNA heteroplasmy before implantation.

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