277 LIPID ACCUMULATION DURING BOVINE OOCYTES Gyr/HOLSTEIN MATURATION COLLECTED BY OPU IN SPOM SYSTEM

2015 ◽  
Vol 27 (1) ◽  
pp. 227
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
C. O. P. Vasconcelos ◽  
...  
Keyword(s):  
Lipid Content ◽  
Lipid Accumulation ◽  
Lipid Analysis ◽  
Fold Increase ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Nuclear Maturation ◽  
Tropical Conditions ◽  
Bovine Oocytes

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. Oocyte quality is crucial for IVP, and lipid accumulation is a detrimental factor. The aim of this study was to assess the effect of SPOM maturation system (Albuz et al. 2010 Hum. Reprod. 25) on lipid accumulation in bovine oocytes. Oocytes obtained by ovum pick-up (OPU) from heifers without ovarian stimulation were transferred to control media (TCM 199 + sodium pyruvate, ITS, penicillin-streptomycin, BSA, FSH, oestrogen, and hCG) or SPOM (2 h pre-IVM: TCM 199 hepes + sodium pyruvate, ITS, penicillin-streptomycin, BSA, IBMX, and forskolin; followed by 28 h IVM: control media + cilostamide) in 5% CO2 atmosphere at 38°C. Oocytes were collected after 20, 24, and 28 h (groups: C20, C24, C28; S20, S24, S28) and evaluated for nuclear maturation using HOECHST (experiment 1, n = 301, 35–62 per group) and lipid content using Oil Red (experiment 2, n = 163, 14–42 per group). Oocytes from 4 replicates were fixed with PFA and stored at 4°C. For Oil Red, all structures were stained and evaluated at the same day. After being washed in 50% ethanol, oocytes were incubated in 0.2% Oil Red O solution for 10 min. Stained area fraction in each oocyte cytoplasm was measured using ImageJ (NIH). Nuclear maturation analysis was performed by Chi-squared test and Lipid analysis by Kruskal-Wallis and Dunn's post-test (P = 0.05). Distinct superscript letters indicate statistical difference between groups. In experiment 1, we detected a reduction in the percentage of matured (MII) oocytes only in S20 group (C20 = 64.28%a, C24 = 74.28%a, C28 = 60.46%a, S20 = 25.8%b, S24 = 59, 01%a, S28 = 74.13%a). In experiment 2, we detected an increase in lipid content in both control and SPOM groups from 20 to 28 h of IVM (S20 = 5.72a ± 4.41, S24 = 15.95bd ± 7.41, S28 = 37.46c ± 8.68, C20 = 8.65a ± 2.58, C24 = 12.14ad ± 8.08, C28 = 18.50b ± 8.09). For SPOM groups, the lipid content increased 2.78-fold from S20 to S24, and 6.54-fold from S20 to S28. In the control group, only in C28 wasa lipid increase detected, 2.13-fold higher than C20 content. Comparing control and SPOM groups at each time point, S28 displayed a 2.02-fold increase in lipid content compared to C28. We concluded that despite SPOM system being effective in maintaining meiotic arrest until 20 h of IVM and forskolin being present during pre-IVM in SPOM groups, Gyr/Holstein crossbreed oocytes obtained by OPU presented a significant increase in lipid content when matured in SPOM system, even higher than observed for the control group. During the last 8 h of IVM we observed a huge lipid accumulation in both systems, suggesting this might be a crucial period.

164 Evaluation of the lipid content of in vitro-matured bovine cumulus - oocyte complexes in the presence of natriuretic peptides A, B, and C types

2019 ◽  
Vol 31 (1) ◽  
pp. 207
Author(s):  
L. Schefer ◽  
K. R. L. Schwarz ◽  
H. Fernandes ◽  
D. M. P. Paschoal ◽  
F. C. C. Castro ◽  
...  
Keyword(s):  
Lipid Content ◽  
Natriuretic Peptides ◽  
Guanosine Monophosphate ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
In Vitro Production ◽  
Significance Level ◽  
Nuclear Maturation ◽  
Bovine Oocytes

One of the difficulties still observed in in vitro production (IVP) of bovine embryos is the lower cryotolerance of such embryos, which has been related to their increased lipid accumulation during culture in the presence of fetal bovine serum (FBS). Previous studies have indicated that the cyclic guanosine monophosphate (cGMP) pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). Synthesis of cGMP can be caused by activation of membrane guanylate cyclase (mGC), also called natriuretic peptide receptors (NPR1 and NPR2), which are activated by natriuretic peptides (NP) A, B, and C types (NPPA, NPPB, and NPPC). The objective of this study was to investigate the influence of supplementation with NP during in vitro maturation (IVM) on lipid content and nuclear maturation of bovine COC. Pools of 25 COC were submitted to IVM in TCM-199 with 0.2mM sodium pyruvate, 10μg mL−1 gentamicide, 0.5μg mL−1 FSH, 10% FBS, and NPPA (10−5 M), NPPB (10−7 M), or NPPC (10−5 M). The control group was matured without NP. After 24h, cumulus cells (CC) were removed and oocytes (OO) were fixed and permeabilized in 4% paraformaldehyde+0.5% Triton for 20min, stained with 10μg mL−1 Hoechst 33342 for 15min and 1μg mL−1 Nile Red for 30min. Then, the OO were placed in 13μL of ProLong (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, covered with a coverslip, and submitted to epifluorescence microscopy to evaluate nuclear maturation (emission 445-450nm and excitation 475-490nm) and lipid content (emission 590nm and excitation 516-560nm). Data for 4 replicates/group were tested for normality of results and homogeneity of variance, and then submitted to ANOVA, followed by Tukey test using a GraphPad Prism software (GraphPad Inc., San Diego, CA, USA), with a significance level of 5%. Nuclear maturation was influenced only by one of the NP added to IVM medium, NPPC, which reduced maturation rate (68.3% metaphase II, MII) compared with the control (82.7% MII; P<0.05). Maturation rates of NPPA (79.5% MII) and NPPB (85.1% MII) did not differ from the control (P>0.05). Activation of mGC by NPPB generated oocytes with lower lipid content (34.02±1.2 IF/μm2) compared with control (36.98±0.7 IF/μm2; P<0.05). Neither NPPA (36.5±1.4 IF/μm2) nor NPPC (39.3±1.4 IF/μm2) was able to reduce the lipid content in oocytes matured in vitro relative to control (P>0.05). Different NP may have different effects on maturation and lipid content in in vitro matured bovine oocytes; NPPB may be favourable for reducing the lipid content of matured bovine oocytes in vitro.

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.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

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.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

289 THE EFFECTS OF MEIOSIS BLOCKING BY CDK INHIBITORS ON THE ACTIVITY OF MATURATION PROMOTING FACTOR, ERK1 AND 2, AND THE DISTRIBUTION OF CYTOPLASMIC ORGANELLES IN IN VITRO-MATURED BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 233
Author(s):  
R. R. D. Maziero ◽  
C. R. F. Guaitolini ◽  
D. M. Paschoal ◽  
A. M. Crespilho ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Cyclin B1 ◽  
Oocyte Quality ◽  
Control Group ◽  
Cdk Inhibitors ◽  
Cortical Granules ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Cytoplasmic Organelles ◽  
Bovine Oocytes

Studies have suggested that the prematuration with meiotic blockers can improve oocyte quality promoting embryonic development; however, its exact effects on cytoplasmic characteristics remain unclear. Thus, this study aimed to evaluate the effects of meiosis block of bovine oocytes with the CDK inhibitors roscovitine (ROS) and butyrolactone (BL-I) on nuclear maturation, expression, and localization of ERK 1 and 2, cyclin B1, and p34cdc2 proteins and the ultrastructure of oocytes. Immature oocytes from the slaughterhouse were divided into the following groups: (1) control, in vitro maturated (IVM) in TCM 199 for 24 h; (2) ROS 12.5 µM; (3) BL-I 50 µM; and (4) ROS (6.25 µM) + BL-I (25 µM) treatment for 6 h followed by IVM in CDK inhibitor-free medium for 18 h. Immature oocytes and IVM oocytes in each group were then fixed stained by immunofluorescence for nuclear visualisation (n = 600), localization, and expression of ERK1 and 2 proteins, cyclin B1 and p34cdc2 protein (n = 350), and prepared for evaluation of the ultrastructure by electron microscopy (n = 100). Data were analysed using the ANOVA test (nuclear visualisation), Student-Newman-Keuls test (expression of ERK1 and 2 proteins, cyclin B1 and p43cdc2) by the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC, USA). At 6 h of IVM, a lower (P < 0.05) percentage of oocytes were at the metaphase I (MI) stage in the control group (C = 18.2 ± 5.4%) compared with other groups and a higher percentage of oocytes were degenerated in the ROS group (16.3 ± 5.6%) compared with other groups (C = 13.6 ± 4.6%, BL-I = 10.0 ± 4.5%, BL-I+ROS = 8.0 ± 5.6%). At 24 h of IVM, higher (P < 0.05) percentages of oocytes were at the MI stage in the control and ROS group (8.3 ± 5.9% and 6.8 ± 6.4%, respectively). There was no difference (P > 0.05) in percentage of metaphase II (MII) oocytes among the groups. Only the ROS group showed lower fluorescence intensity of ERK1 and 2 proteins in the ooplasma (P < 0.05). Immature oocytes showed higher expression of cyclin B1 and p34cdc2 (P < 0.05). There was no difference in the localization of these proteins in the ooplasm and there was no difference in the oocyte ultrastructure (mitochondria, cortical granules, endoplasmic reticulum, microvilli, zona pellucida, lipid granules) among treatments (P > 0.05). The results suggest that a temporary blocking of oocyte maturation by CDK inhibitors affect neither the expression and distribution of MPF components (cyclin B1/cdc2) nor the distribution of cytoplasmic organelles in IVM oocytes. However, the expression of ERK 1 and 2 in mature oocytes may be reduced by pre-IVM with ROS.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

247 EFFECTS OF SILDENAFIL OR SNAP SUPPLEMENTATION, OR BOTH, DURING IN VITRO MATURATION ON LIPID AND REACTIVE OXYGEN SPECIES ACCUMULATION IN BOVINE OOCYTES AND EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 213
Author(s):  
M. Del Collado ◽  
R. C. Botigelli ◽  
K. R. L. Schwarz ◽  
C. Elias ◽  
C. L. V. Leal ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Lipid Accumulation ◽  
Reactive Oxygen ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Oxygen Species ◽  
Lipid Quantification ◽  
Development Rates ◽  
Bovine Oocytes

Previous studies have demonstrated that a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillmaine, SNAP) and a phosphodiesterase inhibitor (Sildenafil, SILD) delay the meiotic resumption of oocytes removed from the follicular environment, and therefore could be used to improve the quality of in vitro-matured (IVM) oocytes. However, it has been reported that SILD-treated cells have increased lipid metabolism and that NO supplementation can modulate the oxidative stress. This study aims to determine the effects of SNAP or SILD supplementation, or both, during IVM on embryo developmental rates, on lipid accumulation of IVM oocytes and on reactive oxygen species (ROS) and lipid accumulation of embryos derived from IVM oocytes. Bovine oocytes were cultured in TCM199 containing 1.0 μg mL–1 of FSH, 50 μg mL–1 of hCG, 1.0 μg mL–1 of oestradiol, 0.2 mM pyruvate, 83.4 μg mL–1 of amikacin, 10% FBS (control group; GCONT), supplemented with 10 µM SILD (GSILD), 0.1 µM SNAP (GSNAP) or both (GS+S). After 24 h of IVM, matured oocytes were assessed for lipid quantification (approximately 49 per group) or used for in vitro embryo production (IVP; approximately 340 oocytes per group). For lipid quantification, denuded oocytes were fixed with 5% triton in 4% paraformaldehyde (PFA) for 30 min and stained with 1 ng mL–1 of Nile Red for 30 min. Embryo lipid analyses (approximately 55 per group) were performed as described for oocytes. For ROS assessment (approximately 58 per group), IVP embryos were stained with 10 µM of H2DFFDA for 1 h and fixed for 30 min in 4% PFA. Stained oocyte and embryo assessments were performed on epifluorescence microscopy, and captured images were analysed on ImageJ (NIH, Bethesda, MD, USA) to quantify the fluorescence intensity (f.i). Statistical analyses were performed with data from 3 replicates for oocytes and 4 for embryos: statistical differences were assessed for lipid and ROS quantity and development rates by split-plot ANOVA. Variables considered in the model were SNAP (presence/absence) and SILD (presence/absence). Means were compared by Student's t at P < 0.05. Regarding oocyte lipid accumulation, groups with SILD (GSILD and GS+S) presented higher lipid quantity (f.i: 52.11 and 47.24, respectively) compared with GCONT and GSNAP (f.i: 38.86 and 41.86, respectively). Supplementation during IVM did not affect development rates (cleavage of 88.1, 88.2, 88.8, and 89.5% and blastocyst rates of 41.2, 38.6, 40, and 41.2% for GCONT, GSNAP, GSILD, and GS+S, respectively). Regarding embryo lipid quantity, similar to oocyte results, SILD groups (GSILD and GS+S) presented higher lipid accumulation (f.i: 68.9 and 68.5, respectively) compared with GCONT (f.i: 55.8) and GSNAP (f.i: 58.9). Considering embryo ROS quantity, GCONT (f.i: 35.9) and GS+S (f.i: 34.2) had the highest levels; however, GS+S did not differ from GSNAP (f.i: 32.85), which was similar to GSILD (f.i: 30.4). In conclusion, SILD had a negative effect on lipid accumulation, which could be due to increased lipid synthesis without increasing lipid oxidation because no increase of embryo ROS levels was observed.

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