scholarly journals 25 MATERNAL-TO-EMBRYONIC TRANSITION FOLLOWING NUCLEAR TRANSFER OR PARTHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 121
Author(s):  
T. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
I. Lagutina ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
De Novo ◽  
Gene Expression Pattern ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Parthenogenetic Activation

The successful development of embryos generated by somatic cell nuclear transfer (SCNT) requires the ooplasm to reprogram the nucleus. This establishes the gene expression pattern necessary for full development by mechanisms that are currently being clarified. The ooplasm action on somatic nuclei shows many common aspects to the process that leads to the creation of a functional embryonic genome from the differentiated sperm and egg genomes. In order to investigate this aspect we studied a critical phase of early embryonic development: the maternal to embryonic transition (MET). We compared the pattern and level of gene expression between bovine embryos derived from in vitro fertilization (IVF), from nuclear transfer of adult fibroblasts (NT), or from parthenogenetic activation (PG). The study was performed in cattle because MET, in this species, occurs over four cell cycles, making it easier to detect even small deviations. Oocytes, matured for 22 h and fertilized in vitro or after cumulus removal, were enucleated and fused to fibroblast cells. Nuclear transfer and Met II oocytes were activated at 24-26 h of maturation with ionomycin (5 �M) for 5 min and 6DMAP (2 mM) for 4 h and then cultured in mSOFaa. Embryos were harvested at the required time for analysis at the 2-, 4-, 8-, and 16-cell; morula; and blastocyst stages and stored snap-frozen in a minimal volume of medium in groups of 5-10 embryos. Semiquantitative RT-PCR was used to study the expression of Nanog, Oct-4, Zar-1, and Par-3, because these genes are directly involved in early embryo development and have a specific expression pattern during MET. Data were analyzed with one-way ANOVA followed by Student-Newman-Keuls All Pairwise Multiple Comparison. No difference in pre-implantation development was observed among the three groups. The Nanog expression pattern was unchanged in all three groups, becoming detectable from the 8-16-cell stage onward. Oct-4 mRNA was detected at all stages in every group, but only in NT embryos did a significant increase occur at the 16-cell stage, suggesting the onset of an anticipated embryonic transcription. the Zar-1 expression pattern, with the characteristic de-novo transcription peak at the 4-cell stage, was observed in both IVF and NT embryos but not in PG embryos. In this group, Zar-1 mRNA levels were significantly higher at the 2- and 4-cell stage than in all of the following stages. The Par-3 gene showed the biggest differences among groups: IVF embryos expressed this gene from the 8-cell stage onward, whereas NT embryos showed high levels of Par-3 mRNA already at the 2-cell stage. Surprisingly, PG embryos showed no detectable Par-3 levels at any stages. The results indicate that, although in vitro development was not affected, gene-specific expression differences during MET occurred among groups. Relating the specific functions exerted by each of these genes in early development to the changes observed following the different manipulations provides useful data toward a better understanding of the role of these genes and of the mechanisms of nuclear reprogramming. This work was supported by FIRB RBNE01HPMX, FIRST 2004, and ESF-EuroStells.

185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 241 ◽  
Author(s):  
H. S. Pedersen ◽  
R. Li ◽  
Y. Liu ◽  
P. Løvendahl ◽  
P. Holm ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Time Interval ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Development Capacity ◽  
Developmental Capacity

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

Patients who failed to conceive following an in vitro fertilization cycle can be clustered into different failure causes using gene expression hierarchical analysis†

2020 ◽  
Vol 103 (3) ◽  
pp. 599-607
Author(s):  
Chloé S Fortin ◽  
Scot Hamilton ◽  
Martin Laforest ◽  
Marie-Claude Léveillé ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Clustering Analysis ◽  
Gene Expression Pattern ◽  
Hierarchical Analysis ◽  
Stimulation Protocol ◽  
Follicular Cells ◽  
Vitro Fertilization ◽  
Different Response

Abstract The patient’s response to an IVF stimulation protocol is highly variable and thus difficult to predict. When a cycle fails, there are often no apparent or obvious reasons to explain the failure. Having clues on what went wrong during stimulation could serve as a basis to improve and personalize the next protocol. This exploratory study aimed to investigate if it is possible to distinguish different failure causes or different follicular responses in a population of nonpregnant IVF patients. Using qRT-PCR, we analyzed a panel of genes indicative of different failure causes in patients who did not achieve pregnancy following an IVF cycle. For each patient, a pool of follicular cells from all aspirated follicles was used as a sample which gives a global picture of the patient’s ovary and not a specific picture of each follicle. We performed hierarchical clustering analysis to split the patients according to the gene expression pattern. Hierarchical analysis showed that the population of nonpregnant IVF patients could be divided into three clusters. Gene expression was significantly different, and each cluster displayed a particular gene expression pattern. Follicular cells from patients in clusters 1, 2 and 3 displayed respectively a pattern of gene expression related to large incompetent follicles with a higher apoptosis (over matured), to follicles not ready to ovulate (under mature) and to an excess of inflammation with no visible symptoms. This study reinforces the idea that women often have different response to the same protocol and would benefit from more personalized treatments.

Acute Myeloid Leukemia with Translocation t(8;16) Demonstrates Specific Cytomorphological, Cytogenetic, and Gene Expression Characteristics and Can Clearly Be Discriminated from Other AML with Balanced Translocations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2897-2897
Author(s):  
Torsten Haferlach ◽  
Helmut Loeffler ◽  
Alexander Kohlmann ◽  
Martin Dugas ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Who Classification ◽  
De Novo ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Support Vector ◽  
Gene Expression Patterns ◽  
Down Regulation ◽  
Specific Expression ◽  
Balanced Translocations

Abstract Balanced chromosomal rearrangements leading to fusion genes on the molecular level define distinct biological subsets in AML. The four balanced rearrangements (t(15;17), t(8;21), inv(16), and 11q23/MLL) show a close correlation to cytomorphology and gene expression patterns. We here focused on seven AML with t(8;16)(p11;p13). This translocation is rare (7/3515 cases in own cohort). It is more frequently found in therapy-related AML than in de novo AML (3/258 t-AML, and 4/3287 de novo, p=0.0003). Cytomorphologically, AML with t(8;16) is characterized by striking features: In all 7 cases the positivity for myeloperoxidase on bone marrow smears was >70% and intriguingly, in parallel >80% of blast cells stained strongly positive for non-specific esterase (NSE) in all cases. Thus, these cases can not be classified according to FAB categories. These data suggest that AML-t(8;16) arise from a very early stem cell with both myeloid and monoblastic potential. Furthermore, we detected erythrophagocytosis in 6/7 cases that was described as specific feature in AML with t(8;16). Four pts. had chromosomal aberrations in addition to t(8;16), 3 of these were t-AML all showing aberrations of 7q. Survival was poor with 0, 1, 1, 2, 20 and 18+ (after alloBMT) mo., one lost to follow-up, respectively. We then analyzed gene expression patterns in 4 cases (Affymetrix U133A+B). First we compared t(8;16) AML with 46 AML FAB M1, 41 M4, 9 M5a, and 16 M5b, all with normal karyotype. Hierachical clustering and principal component analyses (PCA) revealed that t(8;16) AML were intercalating with FAB M4 and M5b and did not cluster near to M1. Thus, monocytic characteristics influence the gene expression pattern stronger than myeloid. Next we compared the t(8;16) AML with the 4 other balanced subtypes according to the WHO classification (t(15;17): 43; t(8;21): 40; inv(16): 49; 11q23/MLL-rearrangements: 50). Using support vector machines the overall accuracy for correct subgroup assignment was 97.3% (10-fold CV), and 96.8% (2/3 training and 1/3 test set, 100 runs). In PCA and hierarchical cluster analysis the t(8;16) were grouped in the vicinity of the 11q23 cases. However, in a pairwise comparison these two subgroups could be discriminated with an accuracy of 94.4% (10-fold CV). Genes with a specific expression in AML-t(8;16) were further investigated in pathway analyses (Ingenuity). 15 of the top 100 genes associated with AML-t(8;16) were involved in the CMYC-pathway with up regulation of BCOR, COXB5, CDK10, FLI1, HNRPA2B1, NSEP1, PDIP38, RAD50, SUPT5H, TLR2 and USP33, and down regulation of ERG, GATA2, NCOR2 and RPS20. CEBP beta, known to play a role in myelomonocytic differentiation, was also up-regulated in t(8;16)-AML. Ten additional genes out of the 100 top differentially expressed genes were also involved in this pathway with up-regulation of DDB2, HIST1H3D, NSAP1, PTPNS1, RAN, USP4, TRIM8, ZNF278 and down regulation of KIT and MBD2. In conclusion, AML with t(8;16) is a specific subtype of AML with unique characteristics in morphology and gene expression patterns. It is more frequently found in t-AML, outcome is inferior in comparison to other AML with balanced translocations. Due to its unique features, it is a candidate for inclusion into the WHO classification as a specific entity.

56CARDIOMYOCYTES FOR THE STUDY OF DEDIFFERENTIATION IN BOVINE NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 150
Author(s):  
A. Lucas-Hahn ◽  
M. Schwarzer ◽  
E. Lemme ◽  
L. Schindler ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Fusion Rate ◽  
Marker Genes ◽  
Cardiac Marker ◽  
Reconstructed Embryos

Nuclear transfer facilitates the study of the dedifferentiation process of differentiated somatic cells. Cardiomyocytes are a good model of terminally differentiated cells showing a unique gene expression pattern of cardiac marker genes. The purpose of this study was to test bovine cardiomyocytes as donor cells in nuclear transfer. Cardiomyocytes were isolated from fetal heart muscle (3–5 months of gestation), which were obtained at the abbatoir and immediately perfused with cold Custodiol (Dr. Franz Köhler Chemie, Germany) to reduce metabolism and protect the cells against ischaemia. Subsequently, hearts were perfused with collagenase in Krebs-Henseleit buffer (KHB) to dissociate the tissue and isolate single elongated, contractile cells. For nuclear transfer and fusion the cardiomyocytes were rounded up by exposure to increasing calcium concentrations (2.5–200μM) in the culture medium before the cells were incubated in suspension for 46–48 hours in MEM medium plus 10% FCS. Nuclear transfer was performed as described earlier (Lucas-Hahn et al., 2002, Theriogenology 57, 433). As a control, adult female fibroblasts were employed. Fusion rate, cleavage (day 3 of in vitro culture) and development up to the morula/blastocyst (day 7 of in vitro culture) were recorded and statistically analysed with Student’s t-test. A total of 243 nuclear transfer complexes with cardiomyocytes and 127 with fibroblasts were produced. Fusion rates for cardiomyocyte complexes were significantly (P<0.001) lower (28.8%) compared to fibroblasts (84.3%). Cleavage rates were 48.1% for cardiomyocytes and 62.8% for the fibroblast-derived embryos. The developmental capacity to the morula/blastocyst was significantly (P<0.01) reduced for cardiomyocyte (9.4%) compared with the fibroblast-derived (32.4%) reconstructed embryos. Most of the Day 7 embryos were frozen for investigation of gene expression patterns of cardiac marker genes. Staining with Hoechst 33342 for counting total cell numbers revealed that 87.3±20.9 blastocysts were derived from fibroblasts and 100 blastocysts from cardiomyocytes. These results indicate that nuclear transfer with terminally differentiated cardiomyocytes is possible, although with reduced rates. Studies are underway to analyze the gene expression of cardiac marker genes in reconstructed embryos to gain insight into dedifferentiation after nuclear transfer using cardiomyocytes as a model. This study was supported by Deutsche Forschungsgemeinschaft (DFG; Ni 256/16-1)

48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
R. Cancian ◽  
M. Macelai ◽  
G. Tavares ◽  
R. S. Valente ◽  
E. S. Caixeta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Cellular Response ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Canonical Pathways

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

Abstract P007: Cardiomyocyte Damage-Released Extracellular S100A1 Protein Promotes Regeneration of Infarcted Myocardium by Modulating Cardiac Fibroblast Function

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
David Rohde ◽  
Gang Qiu ◽  
Nicole Herzog ◽  
Hugo A Katus ◽  
Angelika Bierhaus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Gene Expression Pattern ◽  
Left Ventricular ◽  
Apical Region ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Cardiomyocytes

Background: Similar to heart muscle-specific creatinkinase (CK-MB), S100A1 protein is released from damaged human cardiomyocytes in response to myocardial infarction (MI). Since S100A1-knock out (SKO) mice display rapid post-MI onset of adverse myocardial remodeling and accelerated transition to heart failure, we assessed the hypothesis that ischemia-related release of S100A1 protein modulates myocardial regeneration. Methods and Results: After LAD ligation in C57/B6 mice, S100A1 serum levels peaked at 10 µg/ml 8 hours post-MI, precisely mirroring the time course previously observed in MI patients. RT-PCR analyses in post-MI whole heart samples revealed significantly lower I-CAM (−50%) and IL-10 (−75%) mRNA abundance as well as heightened Collagen-1 (+40%) and VEGF (+80%) expression in SKO vs. WT mice (p<0.05, n=6 in each group). Interestingly, injection of an S100A1-neutralizing antibody prior to MI in WT mice mimicked the abnormalities observed in post-ischemic SKO animals. To further elucidate extracellular S100A1 biological activity, cardiomyocytes, cardiac fibroblasts (CF), endothelial and smooth muscle cells were exposed to S100A1 in vitro . A rapid internalization of S100A1 was exclusively found in CF, resulting in a phosphorylation of ERK1/2, JNK, and p38 with subsequent activation of NF-kappaB as assessed by Western Blot (WB) and EMSA. RT-PCR and WB analyses revealed significant alterations in CF gene expression in response to S100A1, including an increase in I-CAM (3,5-fold) and IL-10 (20-fold) mRNA levels and diminished Col-1 (−80%) expression. Similar effects were observed after direct injection of S100A1 protein into the left ventricular apical region of WT mice in vivo (S100A1- vs. PBS-injection, n=6). In SKO mice, intraperitoneal application of S100A1 prior to MI largely normalized the adverse gene expression pattern towards WT animals. Conclusions: Our study provides first evidence for cardiomyocyte damage-released S100A1 to act as an endogenous mediator of post-MI inflammation and tissue repair. Considering today's unability to manipulate these molecular mechanisms, extracellular S100A1 might represent a promising target for future therapies of MI.

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