15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer

2021 ◽  
Vol 33 (2) ◽  
pp. 115
Author(s):  
M. Y. Felipe ◽  
M. D. Rodríguez ◽  
L. D. Ratner ◽  
A. De Stéfano ◽  
A. M. Valdez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Transfer ◽  
Geometric Mean ◽  
Internal Standard ◽  
Donor Cell ◽  
Developmental Competence ◽  
Genetic Rescue ◽  
Altered Expression ◽  
Reconstructed Embryos ◽  
Sox2 Gene

Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

357 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF BOVINE OOCYTES AND THEIR SURROUNDING FOLLICULAR CELLS IN SUBJECT TO THEIR DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 335
Author(s):  
H. Torner ◽  
D. Janowski ◽  
N. Ghanem ◽  
D. Salilew-Wondim ◽  
H. Alm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caspase 3 ◽  
Expression Profiles ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Altered Expression ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Bovine Oocytes ◽  
Oocyte Selection

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment. This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).

scholarly journals The Effects of Heat Shock Protein 70 Addition in the Culture Medium on the Development and Quality of In Vitro Produced Heat Shocked Bovine Embryos

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3347
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Medium ◽  
Heat Exposure ◽  
Developmental Competence ◽  
Culture Period ◽  
Negative Effects ◽  
Altered Expression ◽  
Embryo Yield ◽  
Bovine Oocytes

The aims of the present study were to examine the effects of HSP70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n = 1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39 °C throughout the culture period), group C41 (temperature was raised to 41 °C from the 48th to 72nd h post insemination (p.i.) and then it returned at 39 °C for the remaining culture period), and group H41 (the temperature modification was the same as in C41 and during heat exposure, HSP70 was added in the culture medium). Cleavage and embryo yield were assessed 48 h p.i. and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39; and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups. The results of this study confirm the devastating effects of heat stress on embryo development and provide evidence that HSP70 addition at the critical stages can partly counterbalance, without neutralizing, the negative effects of the heat insult on embryos, acting mainly through mechanisms related to energy deployment.

Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer

2010 ◽  
Vol 42 (2) ◽  
pp. 201-218 ◽  
Author(s):  
Dessie Salilew-Wondim ◽  
Michael Hölker ◽  
Franca Rings ◽  
Nasser Ghanem ◽  
Mehmet Ulas-Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrous Cycle ◽  
Embryo Transfer ◽  
Expression Pattern ◽  
Transcriptome Analysis ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Quantitative Alteration

Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30–40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2’-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos

2016 ◽  
Vol 28 (6) ◽  
pp. 824 ◽  
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Developmental Competence ◽  
Simultaneous Treatment ◽  
Altered Expression ◽  
Expression Levels ◽  
Histone 3 ◽  
Donor Cells

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50 nM) and 5azadC (7.5 nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

The Oral Intake of Organic Germanium, Ge-132, Elevates α-Tocopherol Levels in the Plas-ma and Modulates Hepatic Gene Expression Profiles to Promote Immune Activation in Mice

2014 ◽  
Vol 84 (3-4) ◽  
pp. 0183-0195 ◽  
Author(s):  
Takashi Nakamura ◽  
Tomoya Takeda ◽  
Yoshihiko Tokuji
Keyword(s):  
Gene Expression ◽  
Immune Activation ◽  
Expression Profiles ◽  
Water Soluble ◽  
Alpha Tocopherol ◽  
Hepatic Gene Expression ◽  
Tocopherol Concentration ◽  
Altered Expression ◽  
Atp Production ◽  
Transfer Protein

The common water-soluble organic germanium compound poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) exhibits activities related to immune responses and antioxidant induction. In this study, we evaluated the antioxidative effect of dietary Ge-132 in the plasma of mice. Male ICR mice (seven mice per group) received an AIN-76 diet with 0.05 % Ge-132; three groups received the Ge-132-containing diet for 0, 1 or 4 days. The plasma alpha-tocopherol (α-tocopherol) concentration increased from 6.85 to 9.60 μg/ml after 4 days of Ge-132 intake (p < 0.05). We evaluated the changes in hepatic gene expression related to antioxidative activity as well as in the entire expression profile after one day of Ge-132 intake, using DNA microarray technology. We identified 1,220 genes with altered expression levels greater than 1.5-fold (increased or decreased) as a result of Ge-132 intake, and α-tocopherol transfer protein (Ttpa) gene expression was increased 1.62-fold. Immune activation was identified as the category with the most changes (containing 60 Gene Ontology (GO) term biological processes (BPs), 41 genes) via functional clustering analysis of altered gene expression. Ge-132 affected genes in clusters related to ATP production (22 GO term BPs, 21 genes), lipid metabolism (4 GO term BPs, 38 genes) and apoptosis (5 GO term BPs). Many GO term BPs containing these categories were significantly affected by the Ge-132 intake. Oral Ge-132 intake may therefore have increased plasma α-tocopherol levels by up-regulating α-tocopherol transfer protein (Ttpa) gene expression.

scholarly journals Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. O. Olsson ◽  
A. H. Tinson ◽  
N. Al Shamsi ◽  
K. S. Kuhad ◽  
R. Singh ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Large Scale ◽  
Donor Cell ◽  
Blastocyst Formation ◽  
Short Term ◽  
Embryo Formation ◽  
Large Expansion ◽  
Breed Differences ◽  
Breed Comparison

AbstractCloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

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