IGF-I slightly improves nuclear maturation and cleavage rate of bovine oocytes exposed to acute heat shock in vitro

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 514-524 ◽  
Author(s):  
Qi Meiyu ◽  
Di Liu ◽  
Zvi Roth
Keyword(s):  
Growth Factor ◽  
Heat Shock ◽  
Developmental Competence ◽  
Cortical Granule ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Nuclear Maturation ◽  
Igf I ◽  
Bovine Oocytes

SummaryAn in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus–oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

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.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 197
Author(s):  
E. D. Souza ◽  
F. B. E. Paula ◽  
C. C. R. Quintao ◽  
J. H. M. Viana ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
In Vitro Maturation ◽  
Hsp90 Inhibitor ◽  
Stress Conditions ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Cleavage Rate ◽  
Bovine Oocytes

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ingrid Oliveira e Silva ◽  
Danielle Kaiser de Souza ◽  
Flavia Tuany ◽  
Michele Munk Pereira ◽  
...  
Keyword(s):  
Basal Medium ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Serum Free ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Blastocyst Rate ◽  
Defined Culture ◽  
Bovine Oocytes

SummaryThe aim of this study was to evaluate the dose–response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus–oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus–oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

77 EFFECT OF HEAT SHOCK DURING IN VITRO MATURATION ON HETEROCHROMATIN COMPACTION IN BOVINE EMBRYOS AT 4- AND 8-CELL STAGES: PRELIMINARY STUDY

2015 ◽  
Vol 27 (1) ◽  
pp. 132
Author(s):  
L. S. A. Camargo ◽  
T. Aguirre-Lavin ◽  
P. Adenot ◽  
T. D. Araujo ◽  
E. D. Souza ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Gene Activation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Morphological Criteria ◽  
Preliminary Study

High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P > 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P < 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P > 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P < 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P < 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).

184 EFFECT OF HISTONE DEACETYLASE INHIBITOR ON DEVELOPMENT OF EMBRYOS DERIVED FROM HEAT-SHOCKED BOVINE OOCYTES

2017 ◽  
Vol 29 (1) ◽  
pp. 200
Author(s):  
V. R. A. Mendes ◽  
R. B. S. Dias ◽  
J. F. S. Souza ◽  
E. D. Souza ◽  
C. C. R. Quintao ◽  
...  
Keyword(s):  
Heat Shock ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitor ◽  
Negative Impact ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Deacetylase Inhibitor ◽  
Bovine Oocytes

Heat shock affects the oocyte developmental competence and embryonic gene expression. We found that the chromatin organisation of embryos derived from heat-shocked oocytes can also be affected (Camargo et al. 2015 Reprod. Fert. Dev. 27, 132). This study aimed to evaluate whether Scriptaid, a histone deacetylase inhibitor able to modulate the chromatin structure, could influence the development of embryos derived from heat-shocked oocytes. Bovine oocytes were in vitro-matured under conventional temperature (38.5°C) for 24 h (non-heat-shock; NHS group) or under 41.5°C for 12 h followed by 38.5°C for 12 h (heat-shock; HS group). In vitro fertilization was performed under 38.5°C with 5% CO2 in air for 20 h. Right after the end of fertilization the presumptive zygotes from the NHS or HS groups were denuded and randomly exposed to 500 nM Scriptaid for 0, 12, or 24 h, comprising 6 treatments as followa: NHS-0 h (NHS without Scriptaid, n = 185); NHS-12 h (NHS plus Scriptaid for 12 h, n = 178); NHS-24 h (NHS plus Scriptaid for 24 h, n = 177); HS-0 h (HS without Scriptaid, n = 187); HS-12 h (HS plus Scriptaid for 12 h, n = 180); and HS-24 h (HS plus Scriptaid for 24 h, n = 183). After Scriptaid exposure, zygotes were cultured in CR2aa plus 2.5% FCS at 38.5°C with 5% CO2, 5% O2, and 90% N2. Cleavage rate was calculated on Day 2 (44 h post-fertilization) and blastocyst rates on Day 7 and 8 post-fertilization. Six replicates were carried out and data was analysed by logistic regression (Proc Logistic, SAS 9.2, SAS Institute Inc., Cary, NC). Significant data were interpreted as odd ratios considering the 95% confidence interval. Values are shown as mean ± standard error of the mean. There was no difference (P > 0.05) among NHS treatments (NHS-0 h, NHS-12 h, and NHS-24 h) as well as among HS treatments (HS-0 h, HS-12 h, and HS-24 h) for cleavage and blastocyst rates at Day 7. At Day 8, however, the blastocyst rate in the NHS group decreased (P < 0.05) as the time of zygote exposure to Scriptaid increased to 24 h (33.9 ± 2.8 and 24.2 ± 1.6% for NHS-0 h and NHS-24 h, respectively), whereas no difference (P > 0.05) was found in the HS group (20.7 ± 1.5, 21.2 ± 1.6, and 20.5 ± 2.4% for HS-0 h, HS-12 h, and HS-24 h, respectively). Comparison between NHS and HS treatments showed that cleavage and blastocyst rates at Day 7 and 8 of NHS-0 h (88.7 ± 2.8, 30.1 ± 1.5, and 33.9 ± 2.8%, respectively) were superior (P < 0.05) to HS-0 h (79.3 ± 3.2, 16.9 ± 1.0, and 20.7 ± 1.5%, respectively). Differences (P < 0.05) between NHS-12 h and HS-12 h on blastocyst rates at Day 7 (32.8 ± 3.8 v. 20.6 ± 1.7%, respectively) and at Day 8 (31.7 ± 2.7 v. 21.2 ± 1.6%, respectively) were also found. However, no difference (P > 0.05) between NHS-24 h and HS-24 h was found. We showed that Scriptaid for 24 h right after IVF has a negative impact on further development of zygotes derived from oocytes matured under conventional temperature (NHS group), in contrast to zygotes derived from oocytes matured under high temperature (HS group). We concluded that the effect of Scriptaid on embryo development is influenced by the temperature during oocyte maturation. Supported by FAPEMIG, CAPES, and CNPq.

scholarly journals Heat shock during in vitro maturation induces chromatin modifications in the bovine embryo

Reproduction ◽  
2019 ◽  
Vol 158 (4) ◽  
pp. 313-322
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Tiphaine Aguirre-Lavin ◽  
Pierre Adenot ◽  
Thamiris Dornelas Araujo ◽  
Vivian Rachel Araujo Mendes ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Early Embryos ◽  
Further Development ◽  
Bovine Oocytes

Heat stress compromises bovine oocyte developmental competence, but the effects of high temperature during oocyte maturation on embryo chromatin organization is unknown. In this study bovine oocytes were exposed to heat shock (41°C) for 12 h during in vitro maturation and then submitted to in vitro fertilization. The heat shock did not affect (P > 0.05) the cleavage but reduced (P < 0.01) the blastocyst rate on Day 7 and Day 8. No effect (P > 0.05) on total cell number was found, but the heat shock increased (P < 0.05) the proportion of apoptotic cells in blastocysts at Day 8. Immunofluorescence analysis of H3K9me3 and HP1 was performed in embryos at 52 h post in vitro fertilization. An accumulation of H3K9me3 in the nuclei of embryos derived from heat-shocked oocytes at four-cell and eight-cell stages was found. Also, a non-expected higher proportion (P < 0.05) of four-cell stage embryos displaying nuclei with increased HP1 fluorescence was observed, suggesting an abnormal chromatin compaction in embryos from heat-shocked oocytes. Embryos at eight-cell stage derived from heat-shocked oocytes displayed lower (P < 0.05) relative amount of HSP40 transcripts than control ones. In conclusion, heat shock before fertilization has an effect on embryo chromatin, influencing the accumulation of H3K9me3 and HP1 in early embryos as well as further development.

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

scholarly journals Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg
Keyword(s):  
Dna Fragmentation ◽  
Oocyte Maturation ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
The Mean ◽  
Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

Sign in / Sign up

Export Citation Format

Share Document