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 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.

Effects of insulin-like growth factor-I, epidermal growth factor and cysteamine on the in vitro maturation and development of oocytes collected from 6- to 8-week-old Merino lambs

2008 ◽  
Vol 20 (5) ◽  
pp. 570 ◽  
Author(s):  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
W. M. Chis Maxwell ◽  
Simon K. Walker
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Embryo Development ◽  
Insulin Like Growth Factor ◽  
Embryo Viability ◽  
Factor I ◽  
Igf I ◽  
Epidermal Growth ◽  
Growth Factor I

To improve the viability of embryos produced in vitro from lamb oocytes, maturation medium was supplemented with insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), cysteamine, and combinations thereof. Experiment 1 examined the effects of IGF-I supplementation and duration of oocyte maturation on nuclear maturation and embryo development while Experiments 2 and 3 examined the effects of cysteamine and EGF supplementation respectively on embryo development. In Experiment 4, embryo development was examined after maturation with various combinations of supplements. IGF-I supplementation increased cleavage rate (P < 0.05) but its effect on the rate of blastocyst production from original oocytes was variable. Supplementation with IGF-I increased (P < 0.01) the proportion of oocytes at Metaphase II (MII) after 18 h of maturation but not at later times. EGF either alone or combined with IGF-I significantly (P < 0.05) increased cleavage rates compared with other treatment groups but EGF consistently failed to improve blastocyst production rates. Cysteamine improved hatching rates but only when supplemented alone. Maturation of lamb oocytes for 22 h in medium supplemented with 100 ng mL–1 IGF-I and 100 μm cysteamine resulted in the production of 16.0 lambs per donor lamb after embryos were transferred to recipient ewes. It is concluded that EGF and, to a lesser extent, IGF-I, whilst beneficial to initial cleavage, can adversely influence subsequent embryo development. Improvements in embryo viability may more likely be obtained by addressing issues that influence fetal oocyte quality than by modifying in vitro methodology.

scholarly journals Effects of recombinant human follicle-stimulating hormone on embryo development in mice

2005 ◽  
Vol 288 (5) ◽  
pp. E845-E851 ◽  
Author(s):  
L. J. Edwards ◽  
K. L. Kind ◽  
D. T. Armstrong ◽  
J. G. Thompson
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Ovarian Stimulation ◽  
Inner Cell Mass ◽  
Follicle Stimulating Hormone ◽  
Cell Mass ◽  
Nuclear Staining ◽  
Insulin Like Growth Factor ◽  
Stimulating Hormone

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 × CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 ± 5.1%) and 20 (77.3 ± 5.6%) IU rhFSH groups compared with control embryos (96.7 ± 1.0%). The number of inner cell mass cells was reduced ( P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

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