scholarly journals Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

2019 ◽  
Vol 54 (10) ◽  
pp. 1357-1365
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Fernanda Queiros Costa ◽  
Michele Munk ◽  
Sabine Wohlres‐Viana ◽  
Raquel Varela Serapião ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Bovine Embryos

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).

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Rosiara Rosária Dias Maziero ◽  
Carlos Renato de Freitas Guaitolini ◽  
Daniela Martins Paschoal ◽  
André Maciel Crespilho ◽  
Bianca Andriolo Monteiro ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Study Groups ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Maturation Index ◽  
Bovine Oocytes

SummaryThis study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

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.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

167 EFFECT OF DNA METHYLATION INHIBITOR ON HETEROCHROMATIN IN BOVINE EMBRYOS DERIVED FROM HEAT-SHOCKED OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 213
Author(s):  
T. D. Araujo ◽  
J. Jasmin ◽  
C. C. R. Quintao ◽  
E. D. Souza ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Shock ◽  
In Vitro Maturation ◽  
Methylation Status ◽  
Control Groups ◽  
Methylation Inhibitor ◽  
Response To Stress ◽  
Dna Methylation Inhibitor ◽  
And Control

The cell response to stress involves epigenetic modifications in order to regulate the gene expression, which is dependent of chromatin structure and DNA methylation status. On the other hand, changes on DNA methylation can have an effect on chromatin organisation (Espada and Esteller 2010 Semin. Cell. Dev. Biol. 21, 238–246). In this study we evaluated the effect of 5-aza-2′-deoxycytidine (5-aza; Sigma, St. Louis, MO, USA), a DNA methylation inhibitor, on heterochromatin 1 β formation of bovine pre-implantation embryos derived from oocytes that did or did not undergo heat shock during in vitro maturation (IVM). Oocytes were IVM under 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). Oocytes were IVF for 20 h and the denuded presumptive zygotes from NHS or HS groups were cultured with 0 (nontreated controls) or 10 nM of 5-aza for 24 h or 48 h in CR2aa plus 2.5% FCS at 38.5°C with 5% CO2, 5% O2 and 90% N2. Embryos with 4–7 cells at 44 h post-insemination (hpi) and embryos with 8–16 cells at 68 hpi were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β first antibody, then immunofluorescence was evaluated by confocal microscopy (Leica TCS SP5II) and images were processed by ImageJ 1.49 (NIH, Bethesda, MD, USA). Fluorescence of nuclei and of background area (fluorescence/unit area) were measured and then the corrected relative fluorescence per nucleus was calculated. We analysed 129 and 149 nuclei at 44 hpi from 29 and 34 embryos as well as 268 and 182 nuclei at 68 hpi from 37 and 22 embryos of the NHS and HS groups, respectively, obtained from 3 replicates. Data underwent log-transformation and was analysed by ANOVA, and means compared by Student-Newman-Keuls. Embryos with 8–16 cells derived from NHS oocytes and treated with 5-aza for 24 h or for 48 h had nuclei with lower HP1 fluorescence than their respective NHS (nontreated) control (P < 0.01). In contrast, 8–16-cells embryos derived from HS and treated with 5-aza displayed nuclei with the same HP1 fluorescence of their respective HS control (P > 0.05). When embryos derived from HS and NHS (nontreated) control groups were compared, higher HP1 fluorescence was found in those with 4–7 cells of HS group (P < 0.05); however, embryos with 8–16 cells displayed similar HP1 fluorescence between both NHS and HS control groups (P > 0.05). There was no difference on HP1 fluorescence between nuclei of embryos with 4–7 cells treated with 5-aza for 24 h and control (nontreated) in both HS and NHS groups. These data suggest that embryos derived from heat-shocked oocytes can accumulate more heterochromatin at earlier stages than those from non-heat-shocked oocytes and that the effect of DNA methylation inhibition by 5-aza on embryo heterochromatin can vary accordingly to the exposure of the oocyte to heat shock during in vitro maturation. Financial support from CNPq, Fapemig, and CAPES is acknowledged.

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

140 Effect of lycopene supplementation to bovine oocytes exposed to heat shock during in vitro maturation

2022 ◽  
Vol 34 (2) ◽  
pp. 308
Author(s):  
G. Residiwati ◽  
N. Azari-Dolatabad ◽  
H. Tuska ◽  
S. Sidi ◽  
P. Van Damme ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Bovine Oocytes
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