200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 191 ◽  
Author(s):  
F. Poppicht ◽  
H. Stinshoff ◽  
C. Wrenzycki
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Protein Expression ◽  
Mrna Expression ◽  
Early Embryonic Development ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Igf1r Expression

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

70 PROTEIN EXPRESSION OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 149
Author(s):  
F. Poppicht ◽  
H. Stinshoff ◽  
C. Wrenzycki
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Protein Expression ◽  
Early Embryonic Development ◽  
Bovine Embryo ◽  
Liver Protein ◽  
Insulin Like Growth Factor ◽  
Peptide Antibody ◽  
Specific Peptide

Insulin-like growth factor 1 (IGF1) is essential for regulating physiological processes such as growth and development of fetal and placental tissues (Bauer et al. 1998, Fowden 2003). During early embryonic development, IGF1 plays an important role, as it leads to a reduction of apoptosis and decreases early embryonic mortality (Block et al. 2007). The signal transduction of IGF1 is carried out by its specific binding to the membrane-embedded insulin-like growth factor 1 receptor (IGF1R). The expression of IGF1R is a potential quality marker of in vitro produced embryos (Liu et al. 1997, Yaseen et al. 2001). Thus far, analysis of the relative amount of specific transcripts is the method of choice to study bovine pre-implantation embryos, as information on protein expression is scarce. Therefore, it is of great interest to analyse protein expression and to determine if and to which extent these results differ from results obtained in previous mRNA expression analyses. In the present study, a total of 4800 cumulus-oocyte-complexes were deployed in 60 in vitro produced runs. The cleavage rates averaged 57.4 ± 7.3% and blastocyst rates were 27.6 ± 7.5% at Day 8 of culture. Embryos at the blastocyst stage were frozen and stored at –80°C for further experiments. The protein expression of the IGF1R during early embryonic development was investigated by Western blot analysis testing 8 different antibodies. Seven of these antibodies were commercially available and mainly not tested in the bovine species. Only 1 of these antibodies resulted in a weak signal for the IGF1R protein in bovine blastocysts. Therefore, a specific peptide antibody against 2 peptide sequences of the α unit of the bovine IGF1R was produced. The analysis of the IGF1R protein with this antibody resulted in the determination of a signal in a pool of 100 blastocysts, which was weaker than in the positive control (20 μg of bovine liver protein extract). The detection of the IGF1R protein localization was possible in all different stages of embryonic development from the zygote to the expanded blastocyst using immunfluorescence staining with the specific peptide antibody. The IGF1R protein was mainly expressed in the plasma membrane of single blastomeres and also weakly in the cytoplasm. As the early bovine embryo expresses IGF1R throughout all stages, the main function of IGF1 in embryonic development needs to be further elucidated. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

scholarly journals 243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
P. Lonergan ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P.M. Moreira ◽  
B. Pintado ◽  
...  
Keyword(s):  
Growth Factor ◽  
Relative Abundance ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P<0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P<0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

178 IN VITRO CULTURE OF BOVINE EMBRYOS WITH NEUROTROPHINS

2007 ◽  
Vol 19 (1) ◽  
pp. 205
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
C. De Frutos ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
The Other ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Cell Counts

Neurotrophins (NTs) mediate human embryonic stem (hES) cell survival and may also improve methods for hES cell derivation (Pyle et al. 2006 Nature Biotech. 24, 344–350) and quality of the inner cell mass (ICM). We searched published microarray data sets for tyrosine kinase receptors (TRK) (geo data base: GSM27469, GSM27470, GSM27471). The analysis suggested that bovine embryos in vitro at unspecified stages express TRKA, for nerve growth factor (NGF); TRKC, for neurotrophin-3 (NT3); and TRKB, for both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF). NTs functionally cooperate among them and also with basic fibroblast growth factor (bFGF) (Pyle et al. 2006; Logan et al. 2006 Brain 129, 490–502). Experiments in progress include detection of TRK expression by RT-PCR at defined development stages, and analysis of embryonic development with NTs and without bFGF. In this work we cultured embryos matured and fertilized in vitro from slaughterhouse oocytes for 8 days in SOF medium with 6 g L-1 BSA and 2 ng mL-1 bFGF (negative control). Development was monitored and cells were differentially counted in the ICM and trophectoderm (TE) of expanded and hatched blastocysts. NTs were used during the whole culture at 20 ng mL-1 as single (4 experimental groups: NGF, NT3, NT4, and BDNF) or as pooled (1 group) NT compounds. Data (5 replicates; 1403 oocytes) were processed by GLM and Duncan's test, and expressed as LSM � SE (a,b: P < 0.05). At Day 3, no differences were found at the 5- to 8-cell stage, but NT3 and NT4 increased the proportions of embryos at the 8- to 16-cell stage (19.1 � 2.2 and 20.5 � 2.2, respectively, vs. 12.9 � 2.2 to 13.7 � 2.2 within the other groups). On Day 6, NT4 yielded more morulae than controls, BDNF, and NGF (35.3 � 2.7 vs. 26.1 � 2.7, 27.4 � 2.7, and 27.8 � 2.7, respectively), and did not differ from the other groups. NT4 produced more total Day 7 blastocysts than NT3 and BDNF (12.5 � 2.2 vs. 8.1 � 2.2 and 9.9 � 2.2, respectively), whereas there were no differences within medium and expanded blastocysts and Day 8 blastocysts. Proportions of morulae that formed blastocysts were appreciably lower than in concomitant experiments without bFGF. Pooled NTs showed decreased values as compared to some single NTs within the ICM [13.0 � 4.0 vs. 29.1 � 4.6 (NT3) and 24.9 � 4.3 (NGF)], the TE [89.0 � 8.4 vs. 120 � 11.9 (BDNF)], total cells [102.0 � 8.5 vs. 134.0 � 9.9 (NT3), and 140.0 � 12.1 (BDNF)], and tended to differ (P = 0.08) within ICM/total cells [13.1 � 3.1 vs. 21.6 � 3.6 (controls) and 22.2 � 3.6 (NT3)]. Controls differed from BDNF (TE: 88.1 � 9.8 vs. 120.2 � 11.9; total cells: 110.8 � 10.0 vs. 140.0 � 12.1, respectively), and from NT4 for ICM/total cells (21.6 � 3.6 vs. 11.5 � 2.9, respectively). NT4 is likely to exert a role during early embryonic development. However, these blastocysts showed decreased cell counts in the ICM, probably reflected in the pooled NTs group. Targeting proliferation stimuli specifically to the ICM is difficult to get when the ICM is enclosed in the embryo, in contrast with the isolated ICM or the derived stem cells. This work was supported by Grant AGL2005-04479.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

80 DNA METHYLATION OF INSULIN-LIKE GROWTH FACTOR 2 (IGF2) GENE IN DAY 14 IN VITRO-PRODUCED BOVINE EMBRYOS OF DIFFERENT SIZES

2015 ◽  
Vol 27 (1) ◽  
pp. 133
Author(s):  
J. O. Carvalho ◽  
M. M. Franco ◽  
G. M. Machado ◽  
M. A. N. Dode
Keyword(s):  
Dna Methylation ◽  
Growth Factor ◽  
Embryo Development ◽  
Methylation Pattern ◽  
Lower Percentage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Igf2 Gene ◽  
Exon 10

In mammals, a correct DNA methylation reprogramming and the maintenance of genomic imprinting after fertilization are essential for embryo development and pregnancy. One important imprinted gene, related to embryo development and placentation, is the insulin-like growth factor 2 (IGF2) gene. Therefore, embryos with different sizes could show differences in the methylation pattern of IGF2 gene. The aim of this study was to evaluate the methylation pattern of the differentially methylated region (DMR) located within exon 10 of the IGF2 gene, of in vitro-produced Nellore bovine embryos that were different in size on day D14 of development. The embryos were produced from oocytes obtained by follicular aspiration of slaughter house ovaries. On D7 after in vitro fertilization only grade I blastocysts were selected and, in groups of 10 embryos, were transferred non-surgically to the uteri of previously synchronized recipients with similar conditions. Seven days after being transferred, embryos were collected (Day 14 of development) and measured using Motic Images Plus 2.0 program (Motic, Richmond, BC, Canada). Embryos >45 mm were considered large (L) and those <25 mm were considered small (S). After being measured, a portion of each trophoblast layer was biopsied and used to determine the methylation status of the IGF2 gene by bisulfite sequencing. The methylation pattern was evaluated on individual embryos considered as separate replicates. At least 5 to 8 clones were evaluated per embryo and the sequences were analysed with the BiQAnalyser software (Max-Planck-Institut für Informatik, Saarbrücken, Germany), using the GenBank sequence NM_174087.3 as reference. The methylation pattern of the different groups was compared using Kruskal-Wallis test (P < 0.05). No differences in DNA methylation were found between S (26.7 ± 8.3%, n = 37 clones, 5 embryos) and L (34.8 ± 2.9%, n = 20 clones, 4 embryos) embryos. It is already known that the region studied is hypermethylated in sperm and hypomethylated in oocytes and, in some somatic cell types, it is expected to be around 50% methylated, being an imprinted region. Although we found a lower percentage of methylation than that expected for an imprinted region, this pattern may be the physiological pattern for trophoblast cells. This is the first report describing the methylation pattern of this region of the IGF2 gene in Day 14 bovine embryos of different sizes. It can be concluded that the methylation pattern of the intragenic DMR on exon 10 of IGF2 gene of in vitro-produced embryos on Day 14 of development is not affected by embryo size.This work was supported by CNPq, FAP-DF.

scholarly journals The Prognostic Values of the Insulin-Like Growth Factor Binding Protein Family in Ovarian Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Ruoyi Zheng ◽  
Wenming Chen ◽  
Weiting Xia ◽  
Jingyu Zheng ◽  
Qing Zhou
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor ◽  
Protein Expression ◽  
Mrna Expression ◽  
Binding Protein ◽  
Progression Free Survival ◽  
Prognostic Impact ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Factor Binding

Purpose. To assess the expression of insulin-like growth factor binding protein (IGFBP) family and its prognostic impact in ovarian cancer (OC) patients. Materials and Methods. The mRNA expression and protein expression of individual IGFBPs in healthy ovarian samples and OC tissues were explored through Oncomine, Gene Expression Profiling Interactive Analysis, and Human Protein Atlas database. Additionally, the prognostic values of the six IGFBP members in patients with OC were evaluated by Kaplan-Meier plotter. Results. IGFBP2 and IGFBP4 mRNA expression were remarkably upregulated in patients with OC. To be specific, the mRNA expression of IGFBP2 was upregulated in patients with serous ovarian cancer (SOC), while IGFBP1/3/4/5/6 mRNA levels were downregulated. In addition, the IGFBP4 protein expression was upregulated in SOC, and the IGFBP6 protein expression was upregulated in both of SOC and endometrioid ovarian cancer (EOC) tissues. High IGFBP1 mRNA levels showed favorable overall survival (OS) and progression-free survival (PFS) in all OC. Meanwhile, increased IGFBP5/6 mRNA levels revealed worsen OS and PFS in all OC patients. IGFBP4/6 mRNA levels predicted unfavorable OS and PFS only in SOC patients. Moreover, the aberrant mRNA expression of IGFBP1/2/4/5/6 was correlated with significantly prognosis in patients receiving different chemotherapeutic regimens. Conclusion. This study indicates that the IGFBP family reveals distinct prognosis in patients with OC. IGFBP1/2/4/5/6 are useful prognostic predictors for chemotherapeutic effect in OC patients, and IGFBP2/4 are potential tumor markers for the diagnosis of OC.

The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development

2016 ◽  
Vol 28 (4) ◽  
pp. 482 ◽  
Author(s):  
Qi-En Yang ◽  
Manabu Ozawa ◽  
Kun Zhang ◽  
Sally E. Johnson ◽  
Alan D. Ealy
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Embryonic Development ◽  
Embryo Development ◽  
Early Embryonic Development ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Kinase C

Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.

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