45 PROLONGING THE FIRST CELL CYCLE IN NUCLEAR TRANSFER BOVINE EMBRYOS DOES NOT INCREASE CLONING EFFICIENCY

2010 ◽  
Vol 22 (1) ◽  
pp. 180
Author(s):  
R. G. Blaza ◽  
J. E. Oliver ◽  
B. Oback ◽  
D. N. Wells
Keyword(s):  
Cell Cycle ◽  
Embryo Development ◽  
Kinase Inhibitor ◽  
S Phase ◽  
Brdu Incorporation ◽  
Cloning Efficiency ◽  
Cell Stage ◽  
Embryo Survival ◽  
Exact Test

We hypothesized that reprogramming a somatic cell following NT is a time-dependent process that can be improved by artificially prolonging the first cell cycle of the cloned embryo. Eleven candidate drugs were initially screened for their ability to reversibly delay the onset of the first cleavage in bovine parthenotes without affecting subsequent in vitro embryo development. After identifying the cyclin-dependent kinase inhibitor butyrolactone-1 (BLT1; BIOMOL International, Plymouth Meeting, PA, USA) as a suitable candidate, we determined its optimal concentration and exposure time. We then performed zona-free bovine NT with serum-starved male skin fibroblasts. Commencing 10 h after the start of IVC, reconstructed 1-cell embryos were treated with either 200 μM BLT1 or 0.4% DMSO in SOF culture medium for 8 to 11 h. After thorough washing, cleavage rates were recorded and culture continued until Day 7. Labeling with 5-bromo-2-deoxyuridine (BrdU) was used to determine DNA replication during the first cell cycle. Some embryos were also transferred singularly to recipient cows. Embryo development was analyzed by a generalized linear model with binomial variation and pregnancy rates by Fisher’s exact test. At 0, 2, 4, 6, and 10 h after the start of IVC, 0% (0/28), 8% (5/61), 67% (39/58), 90% (54/60), and 100% (16/16), respectively, of NT reconstructs had incorporated BrdU, indicating that all 1-cell NT embryos were in S-phase at the start of treatment. After 8 to 11 h of incubation in BLT1, only 28% (119/429) of NT embryos had cleaved, compared with 93% of DMSO-treated controls (297/319). After removing BLT1 in those embryos arrested at the 1-cell-stage, there was no BrdU incorporation over the subsequent 1 h (0/17), embryos entered mitosis and by 4 h, 90% had cleaved (86/96). Thus, BLT1-arrested embryos were at a post-replicative stage prior to M-phase. Rates of in vitro embryo development on Day 7, from late morula to expanded blastocyst stages, of either grade 1-3 or grade 1-2 quality, in the BLT1 treatment were not different compared with controls (129/275 = 47% v. 151/309 = 49% and 33% v. 33%, respectively). Nuclei counts in expanded blastocysts from the BLT1 treatment were not significantly different than controls (109 v. 121, n = 31). Embryo survival on Day 35 of pregnancy and for calves to 1 month ofage was also not different between BLT1 and control treatments (13/31 = 42% v. 12/29 = 41% and 6% v. 10%, respectively). In conclusion, treating 1-cell NT embryos in S-phase for 8 to 11 h with 200 μM BLT1 arrested embryos in G2 and delayed cleavage by approximately 6 h. Cell cycle arrest was fully reversible after drug withdrawal, with rates of cleavage and in vitro development comparable to that of controls. The prolongation of the first cell cycle in bovine NT embryos using this method did not, however, increase cloning efficiency. Arrest for longer periods, at other stages of the cell cycle, and using alternative reagents may be beneficial.

scholarly journals Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.

1991 ◽  
Vol 115 (5) ◽  
pp. 1275-1282 ◽  
Author(s):  
T Usui ◽  
M Yoshida ◽  
K Abe ◽  
H Osada ◽  
K Isono ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
De Novo ◽  
S Phase ◽  
Continuous Treatment ◽  
Protein Kinase Activity

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

120 EMBRYO SURVIVAL FOLLOWING LIPID-BASED TRANSFECTION OF 1-CELL STAGE BOVINE EMBRYOS WITH SMALL INTERFERING RNA (siRNA) FRAGMENTS AND/OR DNA

2006 ◽  
Vol 18 (2) ◽  
pp. 168 ◽  
Author(s):  
M. Bertolini ◽  
L. R. Bertolini ◽  
S. G. Petkov ◽  
K. R. Madden ◽  
J. D. Murray ◽  
...  
Keyword(s):  
Embryo Development ◽  
Small Interfering Rna ◽  
Blastocyst Stage ◽  
Qualitative Assessment ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Survival ◽  
Interfering Rna ◽  
Higher Doses

The RNA interference (RNAi) technology is a powerful tool for studies in functional genomics. The aim of this study was to evaluate the effects of a cationic lipid-based small interfering RNA (siRNA) and/or DNA delivery to 1-cell-stage bovine embryos on survival to the blastocyst stage. In vitro-produced (IVP) embryos were generated according to Bertolini et al. 2002 (Theriogenology 58, 973), and cloned embryos were produced by the handmade cloning technique (Vajta et al. 2003 Biol. Reprod. 68, 571) using green fluorescent protein (GFP)-expressing fibroblast cells as nuclear donors. Lipofections were performed on zona-free 1-cell-stage IVP embryos at 24–28 h post-fertilization by exposure to 1% (v/v) Lipofectamine 2000 (Invitrogen Co., CA, USA), 0.002% (w/v) GFP plasmid (pEFGP-N1, Clontech Laboratories, CA, USA) and/or various doses of siRNA GFP-specific siRNA oligonucleotide (Invitrogen) or DNA methyltransferase 1 (Dnmt1)-specific siRNA fragments for 60 min at 39°C, according to 5 treatment groups: (1) zona-intact IVP embryos (controls), (2) zona-free control embryos (controls for embryo development after zona removal), (3) embryos treated with GFP + GFP-siRNA at 0, 50, 100, 200, 400, or 800 nm, (4) embryos treated with Dnmt1-siRNA at 0, 50, 100, 250, or 500 nm, and (5) cloned embryos (positive controls for GFP expression). After treatment, embryos were in vitro-cultured in a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256) for 7 days. Cleavage and developmental rates to at least 8-cell and to blastocyst stages were assessed at 48, 96, and 168 h post-fertilization (hpf), respectively. Data were analyzed by the chi-square test. Cleavage rates in embryos treated with higher doses of siRNA were lower than in all other groups (Table 1). Embryo survival to at least 8-cell stage at 48 h, based on cleavage, was similar among all treatments (data not shown), but survival to blastocyst stage was affected by higher doses of GFP- or Dnmt1-siRNA (Table 1). After a qualitative assessment by fluorescence microscopy at 168 hpf, 40 to 63% of GFP-transfected blastocysts showed various levels of fluorescence, irrespective of the siRNA treatments. Fragments of siRNA are known to be short-lived in cultured cells, although we are still uncertain of their behavior and effects in early bovine embryos. We are currently analyzing the effectiveness of the siRNA transfection in the early IVP and clone embryo. In conclusion, liposome transfection of 1-cell-stage embryos did not affect survival and development to the blastocyst stage. However, survival followed an siRNA dose-response effect, with doses higher than 400 nm appearing to be detrimental to embryo development, with a developmental arrest at or close to the embryonic genome activation period. Table 1. Developmental rate of bovine embryos following lipid-based transfection at the 1-cell-stage

Parental diet-induced obesity leads to retarded early mouse embryo development and altered carbohydrate utilisation by the blastocyst

2012 ◽  
Vol 24 (6) ◽  
pp. 804 ◽  
Author(s):  
Natalie K. Binder ◽  
Megan Mitchell ◽  
David K. Gardner
Keyword(s):  
Cell Cycle ◽  
Embryo Development ◽  
Maternal Obesity ◽  
Preimplantation Embryo ◽  
Male Mice ◽  
Cell Stage ◽  
Female Mice ◽  
The Impact ◽  
Paternal Obesity

Maternal obesity results in reproductive complications, whereas the impact of paternal obesity is unclear. In the present study, the effects of parental obesity on preimplantation embryo cell cycle length and carbohydrate utilisation were investigated. Maternal and paternal obesity were assessed independently by deriving zygotes from normal or obese C57BL/6 female mice mated with normal Swiss male mice (maternal obesity), or from normal Swiss female mice mated with normal or obese C57BL/6 male mice (paternal obesity). Zygotes were cultured in vitro and development was then assessed by time-lapse microscopy and metabolism determined using ultramicrofluorescence. Maternal obesity was associated with a significant delay in precompaction cell cycle kinetics from the 1-cell stage. A significant increase in glucose consumption by embryos from obese compared with normal females occurred after compaction, although glycolysis remained unchanged. Similarly, paternal obesity led to significant delays in cell cycle progression during preimplantation embryo development. However, this developmental delay was observed from the second cleavage stage onwards, following embryonic genome activation. Blastocysts from obese males showed disproportionate changes in carbohydrate metabolism, with significantly increased glycolysis. Overall, metabolic changes were not inhibitory to blastocyst formation; however, blastocyst cell numbers were significantly lower when either parent was obese. These data suggest that both maternal and paternal obesity significantly impacts preimplantation embryo physiology.

scholarly journals Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 334
Author(s):  
Ashraf N. Abdalla ◽  
Waleed H. Malki ◽  
Amal Qattan ◽  
Imran Shahid ◽  
Mohammad Akbar Hossain ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Abc Transporters ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Significant Inhibition ◽  
Pik3ca Mutations ◽  
Reversal Effect ◽  
Development Of Resistance

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. Objectives: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA2020 and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. Methods: The PamChip® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. Results: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA2020 with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA2020 with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG1/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G1 phase, p21/p27, and apoptosis in HT29-5FU cells. Conclusion: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA2020 and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.

In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

1994 ◽  
Vol 12 (1) ◽  
pp. 107-118 ◽  
Author(s):  
A Van Bael ◽  
R Huygen ◽  
B Himpens ◽  
C Denef
Keyword(s):  
Cell Cycle ◽  
Cell Population ◽  
Blot Hybridization ◽  
Postnatal Period ◽  
S Phase ◽  
Female Rats ◽  
Mrna Levels ◽  
Dot Blot ◽  
Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

Transcription of the histone H5 gene is not S-phase regulated

1986 ◽  
Vol 6 (2) ◽  
pp. 601-606
Author(s):  
S Dalton ◽  
J R Coleman ◽  
J R Wells
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Northern Blotting ◽  
S Phase ◽  
Erythroid Cells ◽  
Dividing Cells ◽  
Steady State Levels ◽  
Avian Erythroblastosis Virus ◽  
Histone H5

Levels of the tissue-specific linker histone H5 are elevated in mature erythroid cells as compared with levels in dividing cells of the same lineage. We examined levels of H5 mRNA in relation to the cell cycle in early erythroid cells transformed by avian erythroblastosis virus to determine whether the gene for this unusual histone is S-phase regulated. Northern blotting analyses revealed that during the cell cycle steady-state levels of H5 mRNA remained relatively constant in contrast to levels of the major core and H1 mRNAs which increased approximately 15-fold during S phase. In vitro pulse-labeling experiments involving nuclei isolated from synchronized cells at various stages of the cell cycle revealed that transcription of the H5 gene was not initiated at any particular stage of the cell cycle but was constitutive. In contrast, transcription of the H2A gene(s) initiated in early S phase, was present throughout the DNA replicative phase, and was essentially absent in G1 and G2 phases.

scholarly journals Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1647-1653 ◽  
Author(s):  
A Raza ◽  
Y Maheshwari ◽  
HD Preisler
Keyword(s):  
Cell Cycle ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Total Cell ◽  
Acute Nonlymphocytic Leukemia ◽  
Cell Cycle Time ◽  
Myeloid Leukemias ◽  
History Of

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

Stage specificity in the mesenchyme requirement of rodent lung epithelium in vitro: a matter of growth control?

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 183-206
Author(s):  
Kirstie A. Lawson
Keyword(s):  
Cell Cycle ◽  
Bronchial Epithelium ◽  
Branching Morphogenesis ◽  
S Phase ◽  
Growth Fraction ◽  
Early Event ◽  
Rat Lung ◽  
Lung Epithelium ◽  
Dividing Cells

Epithelia from lung rudiments in which secondary bronchial buds are already established (14th and 13th gestational day for rat and mouse respectively) are able to undergo branching morphogenesis and cytodifferentiation in submandibular mesenchyme in vitro, whereas lung epithelium from one day younger foetuses rarely gives a morphogenetic response to submandibular mesenchyme and usually differentiates into primary (non-budding) bronchial epithelium. The failure of 13-day rat lung epithelium to respond to submandibular mesenchyme can be prevented by peeling off the submandibular mesenchyme from the lung epithelium after 2½ days culture and replacing the same mesenchyme, or renewing it with fresh salivary mesenchyme ex vivo. Changes in the epithelial contour are visible by 10 h and buds form within 24 h; this is followed by branching morphogenesis in more than 66% of the samples. The number of cells in S-phase in the epithelium is doubled within 3 to 5 h after the operation and the number of mitotic cells (colchicine block) is increased during an 11 to 19 h period after the operation. Substituting stomach mesenchyme for submandibular mesenchyme after the operation failed to elicit morphogenesis or an increase in the number of S-phase cells in the epithelium. The proportion of epithelial cells in S-phase in unoperated recombinates does not differ from the proportion in the primary bronchial epithelium (non-budding) of homotypic lung recombinates, whereas the proportion of S-phase cells in operated recombinates approaches that found in the buds of homotypic lung recombinates. The distribution of S-phase cells in visibly responding recombinates 15 to 17 h after operation shows the same heterogeneity as in homotypic lung recombinates, newly formed buds having twice as many cells labelled with [3H]thymidine as the non-budding area. Cell cycle parameters of intact rat lung growing in vitro were estimated using the labelled mitoses method. Primary bronchial epithelium and bronchial buds both had a total cell cycle time of about 13 h and an S-phase of about 10 h. The growth fraction was 0·54 in the primary bronchus and 0·95 in the buds. It is suggested that, also in the recombinates, differences in the proportion of S-phase cells at any one time in morphogenetically active and inactive areas of the epithelium are due to differences in the growth fraction. It is concluded that an early event in the morphogenetic response of lung epithelium to submandibular mesenchyme after removing and restoring the mesenchyme is an increase in the size of the population of dividing cells and it is suggested that a high proportion of dividing cells in an epithelial population is a prerequisite for further interaction of epithelium and mesenchyme leading to branching morphogenesis.

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