114 Insulin-Like Growth Factor (IGF)2 and IGF2 Receptor (IGF2R) Murine Blastocyst Transcriptional Response after High Gaseous Pressure Exposure at 8-Cell Stage

2018 ◽  
Vol 30 (1) ◽  
pp. 196
Author(s):  
B. S. Becker ◽  
F. J. F. Collares ◽  
A. V. Gonsiorosky ◽  
C. Rodrigues de Freitas ◽  
D. A. Mentz ◽  
...  
Keyword(s):  
Growth Factor ◽  
Internal Control ◽  
Growth And Development ◽  
Paternal Allele ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Mus Musculus Domesticus ◽  
Cell Stage ◽  
Imprinted Gene

Insulin-like growth factor 2 (IGF2) is a pleiotropic hormone encoded by an imprinted gene expressed in the paternal allele of mammals, and acts in physiological responses including cell proliferation, differentiation, and development. It is mediated through the IGF1R signalling pathway, whereas the IGF2R, a maternally imprinted gene, acts on lysosomal IGF2 degradation. As imprinted genes, both Igf2 and Igf2r expressions are more susceptible to dysregulation by environmental factors. This study aimed to evaluate the effect of exposure of 8-cell-stage murine embryos to 16 MPa of high gaseous pressure (HGP) on the relative Igf2 and Igf2r mRNA abundance in resulting blastocysts following in vitro culture (IVC). Day-3 embryos were recovered from superovulated Mus musculus domesticus females. Eight-cell embryos were exposed to 16 MPa HGP for either 2 h (P1 group) or 4 h (P2 group), with a Control group not exposed to HGP. Immediately after recovery or HGP exposure, embryos were in vitro-cultured for 48 h in mKSOM medium supplemented with 0.4% BSA at 37.5°C, 5% CO2, 5% O2, 90% N2, and saturated humidity. Resulting blastocysts were collected in pools of 10 and stored at –80°C, pending analysis. Following total mRNA extraction, cDNA syntesis and RT-qPCR were performed according to manufacturers. Values were normalized to the internal control Ppia gene. Relative gene expression was calculated using the 2−ΔΔ Ct approach. Blastocyst rates after IVC were compared by the Chi-squared test (P < 0.05), with relative Igf2 and Igf2r expression data and Igf:Igf2r ratio analysed by ANOVA, after log-transformation when needed, with pairwise comparisons done by the Tukey test (P < 0.05). No differences in blastocyst rates after IVC were observed among groups (Control: 94.2%; P1: 95.4%; P2: 94.1%). However, the Igf2 mRNA relative abundances in blastocysts were 6.3- and 4.2-fold lower in P1 (P < 0.01) and P2 (P = 0.07) than in the Control group, respectively. Likewise, the Igf2r relative transcription levels were 6.6- and 2.2-fold down-regulated in blastocysts from the P1 (P < 0.001) and P2 (P < 0.01) groups, respectively, when compared with controls. Although the relative expression for both genes followed a down-regulation pattern in blastocysts exposed to HGP at the 8-cell stage, the Igf2:Igf2r ratio was 1.9-fold lower in blastocysts in the P2 group (P < 0.05) than Controls, which was similar to the P1 group, indicating a potential stress adaptation response for embryo growth and development after exposure to HGP in the P1 group. It is known that cells under certain conditions of stress may halter growth and development as a response to initiate cellular events to maintain viability. Results from this study appear to translate such response process to HGP in both experimental groups. However, as embryo development and the Igf2:Igf2r ratio in embryos were similar between the Control and the P1 group, exposure to 16 MPa HGP for 2 h at the 8-cell-stage embryo does not seem to affect cell signalling to growth and proliferation up to the blastocyst stage.

166 EFFECTS OF LEPTIN AND IGF-1 ON PRE-IMPLANTATION DEVELOPMENT, DNA FRAGMENTATION, AND GENE EXPRESSION OF BOVINE EMBRYOS CULTURED IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 191
Author(s):  
A. Kaya ◽  
H. Sagirkaya ◽  
M. Misirlioglu ◽  
A. Gumen ◽  
E. Memili ◽  
...  
Keyword(s):  
Growth Factor ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Rt Pcr ◽  
Cell Stage ◽  
Group Iii ◽  
Fragmented Dna ◽  
Group I ◽  
Group Ii

Adequate regulatory proteins, growth factors, and hormones in in vitro embryo culture systems are important for improving the quality of embryos to a level similar to that in vivo conditions. The objective of this study was to define the effects of leptin, insulin-like growth factor-1 (IGF-1), and their combination on embryonic development, apoptosis, and expression profiles of a panel of developmentally important genes. Presumptive zygotes (16–18 h post-insemination) were randomly assigned and cultured in control (no supplementation), 5 ng/mL leptin (Group I), 100 ng/mL IGF-1 (Group II), and 5 ng/mL leptin and 100 ng/mL IGF-1 (Group III), all supplemented with 10% FCS on Day 4. On Day 8, the embryos reaching blastocyst stage were randomly either fixed for determination of DNA-fragmented nuclei by using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) or frozen for real-time relative quantitative RT-PCR analysis. The RT-PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70.1), interferon tau (IF-tau), insulin-like growth factor II receptor (IGF-IIr), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). A total of 349, 322, 347, and 360 zygotes were used for the control group and Groups I, II, and III, respectively. Data were analyzed with a randomized complete block design and arcsine square root transformation of the dependent variables consisting of four treatments and six replicates. Cleavage rates were 79.5, 84.2, 87.3, and 82.4% for the control group and Groups I, II, and III, respectively, and only Group II was different from the control (P < 0.05). The percentages of embryos developed beyond the 8–16 cell stage were 44.2, 48.2, 49.0, and 50.7 for the control group and Groups I, II, and III, respectively, and Group III was different from the control (P < 0.05). Percentages of blastocyst development were 26.7, 29.6, 31.5, and 29.8, and the mean blastocyst cell numbers were 96.6, 98.6, 104.4, and 104.1 for the control group and Groups I, II, and III, respectively. The percentage of nuclei with fragmented DNA were 4.2, 3.3, 2.5, and 1.9 for the control group and Groups I, II, and III, respectively. Addition of IGF-1 and/or combination with leptin (Groups II and III) decreased the number of nuclei with fragmented DNA (P < 0.01) as compared to the control group. Although the expression of Glut1, DcIII, and Igf2r did not change among the groups, IF-tau and Dnmt3a were down-regulated in Group II. Hsp70 and IF-tau were up regulated in Group III. Results indicate that addition of IGF-I in culture media improved the cleavage rate; combination with leptin also improved the development rates to 8–16-cell-stage embryos, decreased the TUNEL-positive nuclei, and altered expression of some of the developmentally important genes.

312 EFFECTS OF TRANSFORMING GROWTH FACTOR ALPHA AND INSULIN-LIKE GROWTH FACTOR 1 SUPPLEMENTATION ON IN VITRO MATURATION OF CANINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245
Author(s):  
A. Sato ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
A. Hara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Synergistic Effect ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Germinal Vesicle ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Treatment Groups

Although in vitro maturation (IVM) of oocytes has been successfully established for many species, the efficiency of IVM in canine oocytes is still very low. As growth factors have been shown to promote oocyte maturation in some species, we investigated whether use of transforming growth factor α (TGF-a) and insulin-like growth factor 1 (IGF-1) might overcome the difficulties of achieving meiotic maturation in cultured canine cumulus-oocyte complexes (COC). Ovaries were obtained from bitches at 6 months to 7 years of age by ovariohysterectomy and were sliced repeatedly to release COC. In the first experiment, the COC were cultured at 38.8°C for 48 h in 5% CO2 in air in medium 199 supplemented with either TGF-a (0, 1, 10, or 100 ng mL–1) or IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). In the second experiment, the synergistic effect of TGF-a and IGF-1 was investigated by culturing COC in medium 199 supplemented with both TGF-a (0, 1, 10, or 100 ng mL–1) and IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). At the end of the culture period, the oocytes were denuded of cumulus cells by pipetting with a fine bore glass pipette; the denuded oocytes were then fixed in Carnoy's solution and stained with Hoechst 33342. The nuclear configuration and chromatin morphology of the oocytes were evaluated under confocal laser scanning microscopy. The cells were assigned to 1 of the following meiotic stages: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Data were analysed by ANOVA with Fisher's PLSD test. In experiment 1, no significant difference were observed in the rates of cells maturing to the MI and MII stages, but that in the 10 ng mL–1 of TGF-a group (56.3%) were larger than in the other treatment groups (38.8–51.0%). The frequencies of MII stage cells in the 5, 10, and 50 µg mL–1 of IGF-1 treatment groups (9.8, 13.3, and 12.2%, respectively) were significantly higher than in the 0.5 µg mL–1 of IGF-1 group and the control group (5.3 and 2.2%, respectively). In experiment 2, the frequency of MI and MII cells in the control, 1 ng mL–1 of TGF-a plus 0.5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1, and 100 ng mL–1 of TGF-a plus 50 µg mL–1 of IGF-1 group were 44.1, 36.1, 63.5, 70.8, and 50.8%, respectively. The frequency of MII cells in the control group and the same treatment groups were 2.8, 7.2, 10.4, 15.3, and 10.8%, respectively. Both frequencies in the 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1 group were significantly higher than in the control group. The TGF-a may act in a paracrine fashion on the surrounding granulosa cells, and IGF-1 may play multiple roles in cellular metabolism, proliferation, growth, and differentiation in canine oocyte maturation, as has been reported for many other species. In conclusion, these results demonstrate that a synergistic effect between TGF-a and IGF-1 produces an increased rate of in vitro maturation to the MI and MII stages in canine oocytes.

Imprinting of insulin-like growth factor 2 is modulated during hematopoiesis

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3023-3028 ◽  
Author(s):  
Ian M. Morison ◽  
Michael R. Eccles ◽  
Anthony E. Reeve
Keyword(s):  
Growth Factor ◽  
Mononuclear Cells ◽  
Model Systems ◽  
Paternal Allele ◽  
Parental Origin ◽  
Insulin Like Growth Factor ◽  
Specific Expression ◽  
Peripheral Blood Mononuclear ◽  
Multistep Process

The transcription of insulin-like growth factor 2 (IGF-2) is affected by genomic imprinting, a multistep process through which the parental origin of a gene influences its transcription. The maternal copy of IGF-2 is silenced in most human tissues, but in the choroid plexus and the adult liver both alleles of IGF-2 are expressed. This study shows that though in peripheral blood mononuclear cells IGF-2shows paternal allele-specific expression, in total bone marrow both alleles are transcribed. This modulation of imprinting is not attributable to use of the P1 promoter, because transcription from the P3 promoter occurred from both alleles. These results suggest that transcriptional recognition of the IGF-2 imprint can be modulated during hematopoiesis and may facilitate the development of in vitro model systems to study the transcriptional recognition of a genomic imprint.

255 EXAMINING THE EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I ON THE MATURATION AND DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES EXPOSED TO HEAT SHOCK

2013 ◽  
Vol 25 (1) ◽  
pp. 275
Author(s):  
Q. Meiyu ◽  
Z. Roth
Keyword(s):  
Growth Factor ◽  
Heat Shock ◽  
Control Group ◽  
Cortical Granule ◽  
Insulin Like Growth Factor ◽  
Cell Stage ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
Bovine Oocytes

Insulin-like growth factor-I (IGF-I) has been suggested as a survival factor for pre-implantation bovine embryos exposed to heat shock (HS). Therefore, the aims of the study were 1) to examine the protective effects of IGF-I on the developmental competence of bovine oocytes exposed to HS, particularly the effects on oocyte cytoplasmic and nuclear maturation, and 2) to examine whether IGF-I administration contracts HS-induced apoptosis in bovine oocytes. In vitro maturation/IVF/in vitro-production procedures were performed as described previously by Gendelman and Roth (2012). Briefly, cumulus–oocyte complexes (n = 250 to 300/group; 5 replicates) were matured (TCM-199 with Earle’s salts; 22 h, 5% CO2) at 38.5°C or exposed to HS (41°C) with or without 100 µg of IGF-I (Sigma, St. Louis, MO, USA). Matured oocytes were IVF (18 h, 38.5°C, 5% CO2) and cultured in K simplex optimized medium (38.5°C, 5% CO2, 5% O2) for 8 days. Cleavage rates for 2- and 4-cell-stage embryos were assessed at 42 h post-fertilization. For each experimental group, a subgroup of matured oocytes (n = 50) was examined at the end of maturation for nuclear status (1 µg mL–1 of Hoechst 33342, Sigma), cortical granule migration (fluorescein isothiocyanate-Lens culinaris agglutinin, Sigma) and apoptotic status (TUNEL, Roche, Basel, Switzerland). Data were analysed by one-way ANOVA (JMP-6, SAS Institute Inc., Cary, NC, USA) followed by Student’s t-test. Data are presented as mean ± SE. The proportion of oocytes that cleaved to the 2- to 4-cell-stage embryos was lower in the HS group than in the control group (56.55 ± 4.49% v. 75.6 ± 4.16%, respectively; P < 0.05). Although not significant, IGF-I increased the proportions of heat-stressed oocytes that cleaved to the 2- to 4-cell stage (62.32 ± 4.49% v. 56.55 ± 4.49%, for HS + IGF-I and HS, respectively). Neither maturation at 41.5°C nor IGF-I supplementation had any effect on cortical granule migration because the proportions of oocytes with a type I, type II, and type III cortical granule distribution were similar in the control and HS groups. However, the proportion of oocytes that underwent nuclear maturation (i.e. having a nucleus at the telophase-I or metaphase-II stages) was significantly lower in the HS group than in the control group (P < 0.01), and IGF-I slightly increased their proportion in HS oocytes (nonsignificant). The proportion of TUNEL-positive oocytes tended to be higher in the HS group compared with the control group (47.9 ± 12.2% v. 28.0 ± 12.2%, respectively; P ≤ 0.09), and IGF-I decreased the proportion of TUNEL-positive oocytes in the HS group to a level (27.4 ± 12.2%) similar to that noted in the control group. In summary, exposing bovine oocytes to a physiologically relevant thermal stress impaired their ability to undergo first cleavages, most likely because of alteration in nuclear rather than cytoplasmic maturation. Insulin-like growth factor-I was found to slightly alleviate the deleterious effects of heat shock on bovine oocytes.

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&lt;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&lt;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.

93 THE INVOLVEMENT OF E-CADHERIN IN THERMOPROTECTIVE FUNCTION OF INSULIN-LIKE GROWTH FACTOR-1 IN 4-CELL HAMSTER EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 139
Author(s):  
A. C. Trejo ◽  
I. B. Abad ◽  
V. M. V. Meza ◽  
A. M. Villa ◽  
J. Z. Abad ◽  
...  
Keyword(s):  
Heat Stress ◽  
Growth Factor ◽  
Culture Medium ◽  
Molecular Mechanisms ◽  
Statistical Significance ◽  
Insulin Like Growth Factor ◽  
Cell Stage ◽  
Adhesion Sites ◽  
E Cadherin

Studies have demonstrated that the early pre-implantation embryo is very sensitive to effects of heat stress in vitro. Heat stress reduces the total cell number in blastocysts and increases apoptosis in blastomeres. Insulin-like growth factor-1 (IGF-1) has been widely studied as a thermoprotective agent for its anti-apoptotic actions. Addition of IGF-1 to the culture medium decreases the effects of heat stress on blastocysts but has no effects on 2-cell embryos. Molecular mechanisms by which IGF-1 decreases apoptosis involve activation of the PI3K/Akt pathway. It is also known that adherens junctions contribute to PI3K/AKT activation mediated by the transmembrane glycoprotein E-cadherin, which is involved in Ca2+-dependent cell-cell adhesion. Within 2- to 8-cell embryos, E-cadherin is mainly inactive and has cytoplasmic localization. 6-Dimethylaminopurine (6-DMAP) induces premature cell flattening and E-cadherin redistribution to adhesion sites in 4-cell embryos. The aim of this study was to induce E-cadherin redistribution in 4-cell hamster embryos and evaluate the thermoprotective function of IGF-1 in these embryos. Four-cell embryos were incubated in the presence of 6-DMAP to induce E-cadherin redistribution to adhesion sites and cultured for 24 h under conditions of heat stress and compared with controls without 6-DMAP. Culture medium was supplemented with IGF-1. At the end of culture, developmental stage and rate of apoptosis were determined and analysed by ANOVA using the General Linear Model (GLM) of SAS (SAS Institute Inc., Cary, NC, USA) procedure with statistical significance at P < 0.05. E-Cadherin redistribution induced by 6-DMAP increased development to the 6-cell stage after 24 h (63.57% v. 38.81%, respectively; P < 0.05) and reduced apoptosis (25% v. 33%, respectively; P < 0.05) under heat-stress conditions. In conclusion, we hypothesise a role for E-cadherin-mediated cell flattening in promoting IGF-1-mediated thermoprotection in pre-compact 4-cell hamster embryos. Further studies are required to confirm this link.

Imprinting of insulin-like growth factor 2 is modulated during hematopoiesis

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3023-3028 ◽  
Author(s):  
Ian M. Morison ◽  
Michael R. Eccles ◽  
Anthony E. Reeve
Keyword(s):  
Growth Factor ◽  
Mononuclear Cells ◽  
Model Systems ◽  
Paternal Allele ◽  
Parental Origin ◽  
Insulin Like Growth Factor ◽  
Specific Expression ◽  
Peripheral Blood Mononuclear ◽  
Multistep Process

Abstract The transcription of insulin-like growth factor 2 (IGF-2) is affected by genomic imprinting, a multistep process through which the parental origin of a gene influences its transcription. The maternal copy of IGF-2 is silenced in most human tissues, but in the choroid plexus and the adult liver both alleles of IGF-2 are expressed. This study shows that though in peripheral blood mononuclear cells IGF-2shows paternal allele-specific expression, in total bone marrow both alleles are transcribed. This modulation of imprinting is not attributable to use of the P1 promoter, because transcription from the P3 promoter occurred from both alleles. These results suggest that transcriptional recognition of the IGF-2 imprint can be modulated during hematopoiesis and may facilitate the development of in vitro model systems to study the transcriptional recognition of a genomic imprint.

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).

DNA Methylation of the Insulin-Like Growth Factor 2-Imprinted Gene in Trophoblast Cells of Elongated Bovine Embryo: Effects of the In Vitro Culture

2019 ◽  
Vol 21 (5) ◽  
pp. 260-269
Author(s):  
Anelise dos Santos Mendonça ◽  
Maurício Machaim Franco ◽  
José de Oliveira Carvalho ◽  
Grazieli Marinheiro Machado ◽  
Margot Alves Nunes Dode
Keyword(s):  
Dna Methylation ◽  
Growth Factor ◽  
In Vitro Culture ◽  
Bovine Embryo ◽  
Insulin Like Growth Factor ◽  
Trophoblast Cells ◽  
Imprinted Gene

scholarly journals 48EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 SUPPLEMENT TO NCSU-23 MEDIUM ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 146
Author(s):  
S. Kim ◽  
D.H. Nam ◽  
Y.W. Jung ◽  
H.S. Kim ◽  
S.H. Lee ◽  
...  
Keyword(s):  
Growth Factor ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Total Cell ◽  
Insulin Like Growth Factor ◽  
Total Cell Number ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Igf I

The developmental potential of in vitro production of embryos is affected by various factors, including the culture system, oocyte quality, the presence of serum, and embryo paracrine and autocrine growth factors. Insulin-like growth factor is a good stimulator of oocyte maturation and embryo development. The present study investigated the effect of insulin-like growth factor-I (IGF-I) supplement on the preimplantation development of porcine embryos derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. The number of total cells and inner cell mass (ICM) cells in blastocysts were counted after differential staining at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). In Experiment 1, a total of 2,462 in vitro-matured oocytes (527, 458, 498, 481 and 498, respectively) were inseminated with frozen-thawed boar semen and subsequently cultured in North Carolina State University (NCSU)-23 medium supplemented with various concentrations of IGF-1 (0, 1, 10, 50 and 100ngmL−1). As a result, significant model effects on the development to the 2-cell stage (P=0.033) and to the blastocyst stage (P=0.0067) were found, and more blastocysts (16.9, 16.6, 17.5, 21.8 and 14.7 %, respectively) were obtained in medium supplemented with 50ngmL−1 of IGF-I. Moreover, increase in the total cell number (56.5, 53.2, 74.0, 76.4 and 58.4) and ICM (6.6, 5.8, 9.3, 9.4 and 6.1) cells was observed in IVF embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 IGF-1. In Experiment 2, porcine cloned embryos were produced by our standard protocol using fetal fibroblasts as donor cells (Hyun SH et al., 2003 Theriogenology 59, 1641–1649) and cultured in NCSU-23 supplemented with the same concentration of IGF-1 as Experiment 1. As a result, a total of 501 reconstructed oocytes (99, 98, 102, 99 and 96, respectively) were cultured and significant model effects on the development to the 2-cell stage (P=0.0179) were found. More blastocysts (10.5, 11.2, 11.8, 20.8 and 10.1%) were produced when embryos were cultured in NCSU-23 medium supplemented with 50ngmL−1, even though no statistical significance was found (P=0.1182). Increases in the total cell number (42.7, 46.0, 45.9, 51.1 and 38.2) and ICM cells (3.8, 3.8, 5.6, 6.6 and 4.8, respectively) were observed in cloned embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 of IGF-I. In conclusion, the present study demonstrated that IGF-1 at the concentration of 50ngmL−1 improves the development of preimplantion embryos derived from IVF and SCNT. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).

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