30 EFFECT OF THE CYTOPLAST SOURCE AND KARYOPLAST TYPE ON THE DEVELOPMENT OF HANDMADE CLONED EMBRYOS IN GOATS

2012 ◽  
Vol 24 (1) ◽  
pp. 127 ◽  
Author(s):  
C. Feltrin ◽  
N. Mohamad-Fauzi ◽  
S. Gaudencio Neto ◽  
L. T. Martins ◽  
J. L. Almeida ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Size ◽  
Polar Body ◽  
Primary Cultures ◽  
Cumulus Cell ◽  
Donor Cell ◽  
Transgenic Goats ◽  
Handmade Cloning

The aim of this study was to compare the effect of 2 donor cell types (bone marrow-derived mesenchymal stem cells, BM-MSC and skin fibroblast cells, SFC) and the source of oocytes (in vivo- and in vitro-matured goat oocytes) on the developmental capacity of handmade cloned goat embryos, following our procedures adapted from cattle (Ribeiro et al., 2009, Cloning Stem Cells 11, 377–386). In vivo- and in vitro-matured oocytes obtained postmortem from 36 superovulated and 90 nonstimulated goats were used as cytoplasts for cloning, after 26 h from the induction of ovulation or 20 h from the onset of IVM, respectively. Subsequent to cumulus cell removal and polar body selection, a total of 242 in vivo- and 580 in vitro-matured oocytes were subjected to zona removal, bisection in cytochalasin B and screening under ultraviolet light. Enucleated hemi-cytoplasts were exposed to phytohemoagglutinin and adhered to a single somatic cell (BM-MSC or SF) and electrofused by two 1.2 kV cm–1 DC pulses for 20 μs. Cell primary cultures were established from lysozyme transgenic goats. Prior to cloning, cells between the 3rd and 8th passage and at 50 to 60% (BM-MSC) or >95% (SFC) confluence were evaluated for size and viability using the CountessTM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). Fused structures were activated in ionomycin/6-DMAP and in vitro-cultured in the well of the well system in SOFaa + 5% FCS + 0.2% BSA, at 38.5°C, in 5% CO2, 5% O2 and 90% N2, for 6 days. After 8 replications, fusion, cleavage (Day 2) and embryo developmental (Day 6) rates were compared by the χ2 test. Data obtained on cell size and viability were analysed by ANOVA (P < 0.05). Cell viability was similar between SFC (86.7 ± 2.2%) and BM-MSC (89.0 ± 2.2%). However, mean cell size was significantly smaller in SFC (14.4 ± 0.4 μm) than in BM-MSC (20.1 ± 0.4 μm). Cell size appeared to be associated with fusion efficiency because fusion rates were also significantly lower with SFC than with BM-MSC (Table 1). However, cell type or oocyte source did not affect any other parameter for embryo production by cloning between groups. A total of 63 compact morulas and blastocysts from both cell and oocyte types were transferred, in groups of 4 to 5 embryos, to 15 synchronous recipients. Pregnancy diagnosis is performed by ultrasonography on Days 28 to 32. Thus far, one pregnancy derived from an embryo reconstructed with in vivo-matured oocytes and BM-MSC was obtained out of 9 recipients that received 37 embryos from all treatment groups. Six recipients with 26 embryos transferred are still pending diagnosis. In conclusion, the handmade cloning procedure using in vivo- and in vitro-matured oocytes and BM-MSC and SFC appears to be an effective alternative for the production of transgenic goats. Table 1.In vitro development of goat embryos produced by handmade cloning using human lysozyme (hLZ) transgenic cell lines Funded by the RECODISA Project, FINEP/MCT/Brazil.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning

2018 ◽  
Vol 30 (1) ◽  
pp. 158
Author(s):  
R. Olivera ◽  
L. Moro ◽  
R. Jordan ◽  
C. Luzzani ◽  
S. Miriuka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Donor Cell ◽  
Control Group ◽  
Nuclear Reprogramming ◽  
Marrow Mesenchymal Stem Cells

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P < 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P < 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P < 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.

scholarly journals In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain

2021 ◽  
Author(s):  
Tyson J Ruetz ◽  
Chloe M Kashiwagi ◽  
Bhek Morton ◽  
Robin W Yeo ◽  
Dena S Leeman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Stem Cells ◽  
Primary Cultures ◽  
Mammalian Brain ◽  
Aging Brain ◽  
Gene Knockouts ◽  
Old Mice

Aging impairs the ability of neural stem cells to transition from quiescence to activation (proliferation) in the adult mammalian brain. Neural stem cell (NSC) functional decline results in decreased production of new neurons and defective regeneration upon injury during aging, and this is exacerbated in Alzheimer's disease. Many genes are upregulated with age in NSCs, and the knockout of some of these boosts old NSC activation and rejuvenates aspects of old brain function. But systematic functional testing of genes in old NSCs - and more generally in old cells - has not been done. This has been a major limiting factor in identifying the most promising rejuvenation interventions. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screening pipeline in primary cultures of young and old NSCs identifies over 300 gene knockouts that specifically restore old NSC activation. Interestingly, the top gene knockouts are involved in glucose import, cilium organization and ribonucleoprotein structures. To determine which gene knockouts have a rejuvenating effect for the aging brain, we establish a scalable CRISPR-Cas9 screening platform in vivo in old mice. Of the 50 gene knockouts we tested in vivo, 23 boost old NSC activation and production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes for the GLUT4 glucose transporter, is a top rejuvenating intervention for old NSCs. GLUT4 protein expression increases in the stem cell niche during aging, and we show that old NSCs indeed uptake ~2-fold more glucose than their young counterparts. Transient glucose starvation increases the ability of old NSCs to activate, which is not further improved by knockout of Slc2a4/GLUT4. Together, these results indicate that a shift in glucose uptake contributes to the decline in NSC activation with age, but that it can be reversed by genetic or external interventions. Importantly, our work provides scalable platforms to systematically identify genetic interventions that boost old NSC function, including in vivo in old brains, with important implications for regenerative and cognitive decline during aging.

43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 140
Author(s):  
N. Hornen ◽  
W. A. Kues ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
P. Hassel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Stem Cell Population ◽  
High Density Culture ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Normal Offspring

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (&lt;14 �m, 15–20 �m, &gt;21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.

scholarly journals 5-Azacytidine depletes hematopoietic stem cells and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice

2021 ◽  
Author(s):  
Andriyana K. Bankova ◽  
Wendy Pang ◽  
Brenda Josefina Velasco ◽  
Janel R. Long-Boyle ◽  
Judith A. Shizuru
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cell Transplantation ◽  
Apoptotic Cell ◽  
Donor Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Immunocompetent Mice

Depletion of hematopoietic stem cells (HSC) is used therapeutically in many malignant and non-malignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSC allowing donor HSC to engraft. Current treatments to achieve HSC elimination rely on modalities that cause DNA strand breakage (i.e., alkylators, radiation) resulting in multiple short-term and long-term toxicities, and sometimes even death. These risks have severely limited HCT utilization to patients with few to no co-morbidities, and excluded many others with diseases curable by HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSC. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSC in vitro. When used to pretreat recipient mice for transplant, AZA permitted low level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb), targeting CD117 (c-Kit), a molecule expressed on HSC, more robust HSC-depletion and substantially higher levels of multilineage donor cell engraftment was achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in HSPC. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its utilization in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSC which can be rapidly tested in the clinical setting.

24 Evaluation of Latrunculin A for the Activation of Hand-Made Cloning (HMC) Porcine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 151
Author(s):  
F. K. Castañeda ◽  
N. G. Canel ◽  
G. V. Landschoot ◽  
A. De Stéfano ◽  
R. J. Bevacqua ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Uv Light ◽  
Electric Pulse ◽  
Polar Body ◽  
Primary Cultures ◽  
Donor Cell ◽  
Activation Process ◽  
Latrunculin A ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) is an important biotechnological tool. However, production rates of viable offspring remain low. One possible cause of this low efficiency is chromosomal losses during early activation process (Liu et al. 2015 Cell. Reprogram. 17, 463–471). The use of actin inhibitors that block second polar body extrusion during activation protocols might be a strategy to avoid such losses. The objective of this work was to compare the efficiency of the use of 2 actin inhibitors during the activation of hand-made cloning (HMC) porcine embryos. One of the compounds used was latrunculin A (LatA), which joins directly to actin monomers, preventing their assembly to the filaments. The other was cytochalasin B (CB), which is commonly used for activation protocols. It binds to the growing actin filaments and prevents their elongation. For this purpose, in vitro-matured cumulus–oocyte compexes were deprived of their cumulus and zonae pellucidae cells by mechanical and enzymatic treatments. Oocytes were randomly distributed in 2 experimental groups (HMC) and 2 parthenogenetic control groups (PA). For HMC groups, oocytes were bisected using a microblade and the resulting hemioocytes were stained with Hoechst 33342 and observed under UV light to identify those that had lost the metaphase II plate. Adult skin fibroblasts from primary cultures were used as nuclear donors. For nuclear transfer, 2 hemicytoplasts were fused to a donor cell by an electric pulse of 1.42 kV/cm for 30 μs. After 2 h of nuclear reprogramming, the reconstituted embryos were activated by an electric pulse of 1.2 kV/cm for 80 μs and incubated with cycloheximide (CHX, 10 μg mL−1 , 3 h) in combination with one of the actin inhibitors: LatA 2 μM (CHX-LatA goup) or CB 2.5 μg mL−1 (CHX-CB group). The PA groups were subjected to the same activation treatments (PA-CHX+LatA and PA-CHX+CB groups). All embryos were cultured in SOFaa medium, using an adaptation of the well-of-the-well (WOW) system (microwells), in a humidified atmosphere with 5% CO2 in air at 39°C. Cleavage, morulae, and blastocysts rates were evaluated at Days 2, 4, and 7-8, respectively. At least 3 replicates were performed per group. Results are presented in Table 1. Our results demonstrate that the production of embryos by HMC activated with CHX-LatA is as efficient as that with CHX-CB, the protocol currently used in SCNT protocols. Further research is needed to study its effect on chromosomal complements and long-term development. Table 1.Effect of activation with cycloheximide (CHX) and latrunculin A (LatA) on in vitro development of hand-made cloning (HMC) porcine embryos (% ± SD in parentheses)

59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 110
Author(s):  
R. Tomii ◽  
B. Ogawa ◽  
H. Nagashima
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Primary Cultures ◽  
Fusion Rate ◽  
Culture Technique ◽  
Domestic Cats ◽  
Donor Cells ◽  
First Polar Body

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.

P–547 Single-cell RNA sequencing identifies molecular regulations associated with poor maturation performance on rescue in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
W T Lee ◽  
K W Ng ◽  
J Liao ◽  
A C S Luk ◽  
H C Suen ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Single Cell ◽  
Rna Sequencing ◽  
Maternal Age ◽  
Polar Body ◽  
Rna Extraction ◽  
Cumulus Cell ◽  
Single Cell Rna Sequencing

Abstract Study question What is the transcriptome signature associated with rescuein vitro matured (rIVM) oocytes? Summary answer GATA–1/CREB1/WNT signaling axis was repressed in rIVM oocytes of poor quality. What is known already rIVM aims to produce mature oocytes (MII) for in vitro fertilization (IVF) through IVM of immature oocytes collected from stimulated ovaries. It is less popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress, and the absence of cumulus cell support in oocytes could account for the failure of rIVM. Study design, size, duration We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo (IVO) oocytes (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors, followed by studying the maternal age effect and ovarian responses on rIVM oocyte transcriptomes. Participants/materials, setting, methods Human oocytes were collected from donors aged 28–41 years with a body mass index of &lt; 30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analyzed. Selected genes in therIVM vs. IVO comparison were validated by quantitative real-time PCR. Main results and the role of chance The transcriptome profiles of rIVM/IVO showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least two-fold change and adjusted p &lt; 0.05) were found to be enriched in metabolic processes, biosynthesis, and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signaling in rIVM when compared with IVO oocytes. We found that estradiol level exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio). rIVM oocytes with higher MII ratio showed over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor outcomes of rIVM, we compared oocytes collected from young donors with high MII ratio versus donors of advanced maternal age and revealed CREB1was an important regulator in rIVM. Our study identified GATA–1/CREB1/WNT signaling was repressed in both rIVM condition and rIVM oocytes of low-quality. Limitations, reasons for caution In the rIVM oocytes of high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle so polar body extrusion was the only indicator. Wider implications of the findings: This study showed that GATA–1/CREB1/WNT signaling and antioxidant actions were repressed in rIVM condition and was further downregulated in rIVM oocytes of low-quality, providing us the foundation of subsequent follow-up research on human subjects. Trial registration number Not applicable

Sign in / Sign up

Export Citation Format

Share Document