scholarly journals Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

2001 ◽  
Vol 53 (2) ◽  
pp. 207-211 ◽  
Author(s):  
M.D. Quetglas ◽  
L.A. Coelho ◽  
J.M. Garcia ◽  
E.B. Oliveira Filho ◽  
C.R. Esper
Keyword(s):  
Growth Factor ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Calf Serum ◽  
Culture Conditions ◽  
Insulin Like Growth Factor ◽  
Chi Square ◽  
Igf I ◽  
Chi Square Test

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

92 INSULIN-LIKE GROWTH FACTOR-1 PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS PRODUCED IN VITRO FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

2016 ◽  
Vol 28 (2) ◽  
pp. 176
Author(s):  
N. A. S. Rocha-Frigoni ◽  
B. C. S. Leão ◽  
P. C. Dall'Acqua ◽  
M. Ambrogi ◽  
G. Z. Mingoti
Keyword(s):  
Oxidative Stress ◽  
Growth Factor ◽  
Molecular Probes ◽  
Insulin Like Growth Factor ◽  
Blastocyst Development ◽  
Chi Square ◽  
Fluorescent Intensity ◽  
Chi Square Test ◽  
Student’S T

The objective of this study was to evaluate the protective effect of insulin-like growth factor (IGF-1) on blastocyst development and cryotolerance of bovine embryos in in vitro culture (IVC) under oxidative stress induced by menadione (MD). Cumulus-oocyte complexes (n = 1421) were matured in TCM-199 with bicarbonate, hormones, and 10% FCS for 22 h. After fertilization, the presumptive zygotes were cultured up to 7 days in SOF medium with 2.5% FCS and 0.5% BSA (control), and also supplemented with 100 μM IGF-1 (IGF). At Day 6, MD was included in the culture medium (0 μM, control; or 5.0 μM, MD) during 24 h. Cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7 (IVF = Day 0). At Day 7, a sample of the blastocysts was stained with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the intracellular ROS levels or was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA). Stained embryos were immediately evaluated under an epifluorescence microscope (excitation 495/550 nm and emission 404/590 nm, respectively, for ROS and TUNEL), and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. Other blastocysts were vitrified (Ingámed®, Maringá-PR, Brazil), and after warming, they were cultured for 24 h to evaluate the re-expansion rates. The results were compared by ANOVA followed by Student’s t-test (mean ± s.e.M) and re-expansion rates by chi-square test (P < 0.05). The cleavage rates did not differ (P > 0.05) among groups (77.1 ± 1.9% to 82.75 ± 2.2%). The blastocyst rates were similar between control (35.4 ± 2.0%) and IGF (34.5 ± 3.7%), and both were higher (P < 0.05) than MD (21.3 ± 2.7%); the IGF+MD group (28.3 ± 1.6%) was similar (P > 0.05) to all groups. The intracellular levels of ROS were higher (P < 0.05) for the MD group (21.7 ± 0.7) than for control (17.0 ± 1.6), and both were similar (P > 0.05) to the IGF (19.2 ± 0.6) and IGF+MD (18.0 ± 1.0) groups. The highest rates of apoptosis were found in the MD group (22.3% ± 2.3) and the smallest in IGF (9.1% ± 0.7), and both differed (P < 0.05) from control (12.8% ± 1.0), and IGF+MD (15.6% ± 1.6). The re-expansion rates were similar between control (77.4%) and IGF (69.2%), and both were higher (P < 0.05) than MD (49.1%); however, the IGF+MD group (57.6%) was similar (P > 0.05) to IGF and MD groups. In conclusion, the supplementation with IGF-1 during IVC reversed the detrimental effects of MD on embryonic levels of ROS and apoptosis, as well as improved the embryo development and cryotolerance of blastocysts under oxidative stress. Financial support was provided by FAPESP (#2012/10083–8 and #2013/07382–6).

scholarly journals 282MATURATION MEDIUM EXCHANGE IS EFFECTIVE ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 261
Author(s):  
Y.S. Park ◽  
S.H. Choi ◽  
H.D. Park ◽  
M.D. Byun
Keyword(s):  
Embryo Development ◽  
Total Protein ◽  
Harmful Effect ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Chi Square ◽  
Medium Exchange ◽  
Chi Square Test

In vitro embryo development is strongly influenced by IVM conditions. Increased duration of IVM may cause aging of the oocytes, which has a harmful effect on the embryo development. Oocyte maturation depends upon the synthesis of several proteins that may play important roles in the cytoplasmic maturation. These experiments were conducted to determine the effect of IVM duration(18-h or 24-h) and medium exchange (at 18h) on embryo development, and to investigate the protein quantities in IVM medium. Korean Native Cow (KNC) ovaries were obtained from a local slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in 50-μL drops of TCM-199 supplemented with 10% fetal calf serum (FBS), 1μgmL−1 MFSH, 10μgmLLH and 1μgmL−1 Estradiol-17β for 18h or 24h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (After Day 3). All types of cultures were carried out in an incubator at 39°C, 5% CO2 in air. The total protein quantity in IVM medium at 18h or 24h were compared by 2-dimensional gel electrophoresis using a 10–15% polyacrylamide gradient gels. Data from three replicates were analyzed by chi-square test. The proportions of oocytes reaching the blastocyst stage was significantly higher in 18h IVM group than 24h IVM group (Table 1). However, there was no difference detected in blastocyst rate between 18h IVM group and 18h medium exchange group. Total protein quantity was reduced between 18h and 24h in IVM medium. There were 299 protein spots identified in IVM medium;; there was an increase at 10 spots in the IVM medium analyzed at 18h and a decrease of 20 spots at 24h. This study suggests that duration of IVM affects subsequent embryo development. The total protein quantity was decreased between 18h and 24h in IVM medium. These proteins may be absorbed into the oocytes and reduce development to the blastocyst stage. However, this may be overcome by IVM medium exchange. Table 1 Effects of duration of IVM and medium exchange on embryo development of KNC oocytes

35 EFFECT OF CULTURE AT LOW OR ATMOSPHERIC OXYGEN TENSION IN SOMATIC DONOR CELLS FOR HORSE NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 165
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlaninan ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Atmospheric Oxygen ◽  
Culture Conditions ◽  
Chi Square ◽  
Reconstructed Embryos ◽  
Donor Cells

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the well system as 3 reconstructed embryos per well. Cleavage and blastocyst formation (7–8 days) of the experimental groups were assessed. In vitro development, on a per-well and RE basis, was compared using the chi-square test. No statistical differences were observed in cleavage [(1): 48/84, 57%; (2): 54/87, 62%). No difference was observed in blastocyst rates on a per-well basis [(1): 5/28, 18%; (2): 4/29, 14%] or on a per-RE basis [(1): 5/84, 6%; (2): 4/87, 5%]. This work suggests that the oxygen tension during the in vitro culture of somatic donor cells does not affect the quantity of the cloned equine blastocyst produced. Further studies are required to determine if these conditions would affect in vivo embryo development.

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 292 MATURATION IN A STRAW IS EFFECTIVE ON THE DEVELOPMENT OF BOVINE OOCYTES IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
Y.M. Park ◽  
S.S. Kim ◽  
J.H. Lee ◽  
Y.S. Park ◽  
H.D. Park
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Development Rate ◽  
Culture Dish ◽  
Chi Square ◽  
Treatment Groups ◽  
Chi Square Test ◽  
Bovine Oocytes

In vitro embryo development is strongly influenced by oocyte maturation environments. Maturation of bovine oocytes is processed in a culture dish. However, the development rate to the transferable blastocyst stage was 10 to 30%. This experiment was to examine the effect of the size of straw and the medium exchange on the development of Korean Native Cow (KNC) oocytes. Ovaries of KNC were obtained from a local slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll-separated spermatozoa (Day 0) in fer-TALP medium for 20 h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). All cultures were maintained in an incubator at 39°C, 5% CO2 in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, we examined the effect of the instrument of maturation (dish or 0.25-mL and 0.5-mL straws) on embryo development. There were no difference in the cleavage (2-cell) among treatment groups. However, the development rate to the 8-cell and blastocyst stage was significantly higher in the 0.5-mL straw (38.5 and 17.0%) than in the 0.25 mL-straw (26.6 and 7.4%, all respectively). In Experiment 2, the KNC oocytes were matured in 0.5-mL straws based on the results of Experiment 1, and we examined the effect of the conditions such as circulation and exchange of maturation medium at 9 h after the start of IVM on embryo development. The development rates to the 2-cell, 8-cell, and blastocyst stage were significantly higher in the circulation group (83.3, 58.0 and 31.3%) than in the control (72.0, 44.7 and 19.3%) and exchange groups (71.3, 40.0, and 18.0%, all respectively). The results of this study suggest that the maturation of KNC oocytes in 0.5-mL straws accompanied by circulation of medium at 9 h is effective in the development to the blastocyst stage.

46 ANTI-APOPTOTIC EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 AND ITS RECEPTOR ON DEVELOPMENT OF PORCINE PRE-IMPLANTATION EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 132
Author(s):  
S. Kim ◽  
S. H. Lee ◽  
J. H. Kim ◽  
Y. W. Jeong ◽  
O. J. Koo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Developmental Competence ◽  
Insulin Like Growth Factor ◽  
Mammalian Embryo ◽  
Vitro Fertilization ◽  
Igf I ◽  
Apoptotic Effect ◽  
Bax Gene

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse pre-implantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/mL), anti-IGF-I receptor (IGR-IR) antibody (0.05 �g/mL), and their combination on porcine pre-implantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both IVF and SCNT embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotropic effect was neutralized by culturing with IGF-I and anti-IGF-I receptor antibody. Significant effects on the development of blastocysts (P < 0.05) were found in IVF (16.9, 22.6, 9.3, and 13.5% for control, IGF-I, anti-IGF-IR antibody, and their combination, respectively) and SCNT (13.2, 21.0, 5.4, and 15.7%) embryos. Culturing IVF and SCNT embryos with IGF-I significantly increased the total number of cells in IVF blastocysts (58.3, 72.4, 41.1, and 55.2; P < 0.05), and SCNT blastocysts (49.2, 60.1, 35.2, and 43.1; P < 0.05), and it decreased the number of apoptotic nuclei in IVF blastocysts (3.9, 2.8, 5.5, and 3.9; P < 0.05) and SCNT blastocysts (4.6, 3.0, 6.1, and 4.9; P < 0.05). These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, whereas expression of the pro-apoptotic Bax gene was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine pre-implantation embryos. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

Longitudinal bone growth in vitro: effects of insulin-like growth factor I and growth hormone

1991 ◽  
Vol 124 (5) ◽  
pp. 602-607 ◽  
Author(s):  
Ben A. A. Scheven ◽  
Nicola J. Hamilton
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Bone Growth ◽  
Long Bone ◽  
Long Bones ◽  
Insulin Like Growth Factor ◽  
Serum Free ◽  
Factor I ◽  
Igf I

Abstract. Longitudinal growth was studied using an in vitro model system of intact rat long bones. Metatarsal bones from 18- and 19-day-old rat fetuses, entirely (18 days) or mainly (19 days) composed of chondrocytes, showed a steady rate of growth and radiolabelled thymidine incorporation for at least 7 days in serum-free media. Addition of recombinant human insulin-like growth factor-I to the culture media resulted in a direct stimulation of the longitudinal growth. Recombinant human growth hormone was also able to stimulate bone growth, although this was generally accomplished after a time lag of more than 2 days. A monoclonal antibody to IGF-I abolished both the IGF-I and GH-stimulated growth. However, the antibody had no effect on the growth of the bone explants in control, serum-free medium. Unlike the fetal long bones, bones from 2-day-old neonatal rats were arrested in their growth after 1-2 days in vitro. The neonatal bones responded to IGF-I and GH in a similar fashion as the fetal bones. Thus in this study in vitro evidence of a direct effect of GH on long bone growth via stimulating local production of IGF by the growth plate chondrocytes is presented. Furthermore, endogenous growth factors, others than IGFs, appear to play a crucial role in the regulation of fetal long bone growth.

Effect of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in monolayer culture

1992 ◽  
Vol 133 (2) ◽  
pp. 291-NP ◽  
Author(s):  
C. Ohlsson ◽  
A. Nilsson ◽  
O. G. P. Isaksson ◽  
A. Lindahl
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Density ◽  
Monolayer Culture ◽  
Culture Conditions ◽  
Factor I ◽  
Sulphate Uptake ◽  
Igf I ◽  
Matrix Production

ABSTRACT The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture. Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 μg/l (60 ± 11% stimulation over control) and for IGF-I at 10 μg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake. In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm2. GH had no significant effect at a low (< 100 000 cells/cm2) or a high (>400 000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm2, but was reduced at higher cell densities (over 250 000 cells/cm2). Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice. The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions. Journal of Endocrinology (1992) 133, 291–300

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.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

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