359 MORPHOLOGIC AND BIOCHEMISTRY ALTERATIONS ON BOVINE OOCYTE MATURATION IN VITRO WITH NITRIC OXIDE AND ITS IMPACT ON EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 336 ◽  
Author(s):  
K. S. Viana ◽  
M. C. C. Bussiere ◽  
C. S. Paes de Carvalho ◽  
B. L. Dias ◽  
M. R. Faes ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Control Group ◽  
Cortical Granule ◽  
Bovine Oocyte ◽  
Cortical Granules ◽  
Maturation In Vitro

The aim of this study was to evaluate morphologic and biochemistry alterations caused by the addition of sodium nitroprusside (SNP), a NO donor, on bovine oocyte maturation in vitro. Bovine ovaries were collected at a local abattoir. COC were cultured in TCM-199 with 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, and antibiotics. Analysis of variance was conducted and the means were compared by t-test at a level of 5%. Experimental design: (1) evaluation of the oocyte plasma membrane viability and integrity using Annexin V/propidium iodide (PI) and Hoechst 33342/PI staining, respectively; (2) microtubule and microfilament organization, and migration of cortical granules by immunofluorescence; (3) oocyte glutathione content and concentration of NO3-/NO2-using the method of Griess (Ricart-Jane D et al. 2002 Nitric Oxide 6, 178-185) and (4) embryo development. In Experiment 1, the addition of 1 mM SNP caused cellular death in the majority of the oocytes [100%, AnnexinV/PI (+) and 80.7% Hoescht/IP (+)] differing from the control group and the 0.01 mM SNP (P < 0.05). In Experiment 2, the microtubule staining was observed in the cytoplasm in both control group and 0.01 mM SNP; however, the group treated with 1 mM of SNP exhibited clear defects in spindle and chromatin arrangements (P < 0.05). No alterations in microfilaments disposition was observed in the control group and 0.01 mM SNP. However, after the addition of 1 mM, the microfilaments arranged into clusters, and not below of the cortex. Oocytes treated with 1 mM SNP (68.2%) showed total cortical granule migration to the periphery of the ooplasm and were similar to the control group (72.2%) (P > 0.05). Nevertheless, in the group treated with 0.01 mM SNP the total cortical granule migration was greater (86.8%, P < 0.05). In Experiment 3, the glutathione content of oocytes cultured in the presence of 1 mM SNP was lower (4.4p mol) when compared to the control group (5.4p mol) and 0.01 mM SNP (5.5 pmol) (P > 0.05). The concentration of NO in the medium were similar to both control group (6.0 ± 3.0 μM) and treated with 0.01 mM SNP (15.8 ± 1.9 μM), however, the treatment with 1 mM SNP increased 10 times (59.9 ± 12.0 μM; P < 0.05) this concentration. In Experiment 4, cleavage rates and embryo development were similar for groups control and 0.01 mM SNP (P > 0.05). Even so, in the group treated with 0.01 mM there was a greater blastocyst cell number when compared to the control group (256.8 ± 52.5 and 196.9 ± 54.0, respectively-P < 0.05). These results indicate that: (1) the addition of 0.01 mM SNP increased the quality of the oocyte maturation, leading to a higher percentage of cortical granules migration and blastocyst cell numbers, in a different pathway from that of glutathione; (2) the addition of 1 mM of SNP caused a cytotoxic effect, leading to cellular death with loss of viability and integrity of plasma membrane, absence of nuclear maturation/organization of cytoskeleton and reduction of the glutathione content, although with no intervention in the migration of cortical granules.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Tamana Rostami ◽  
Fardin Fathi ◽  
Vahideh Assadollahi ◽  
Javad Hosseini ◽  
Mohamad Bagher Khadem Erfan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Control Group ◽  
Vitro Fertilization ◽  
Maturation In Vitro

Summary The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

scholarly journals Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽  
2001 ◽  
pp. 51-75 ◽  
Author(s):  
A Trounson ◽  
C Anderiesz ◽  
G Jones
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Growth Phase ◽  
Developmental Competence ◽  
Minimum Size ◽  
Success Rates ◽  
Human Oocytes ◽  
Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Protein synthesis and phosphorylation during bovine oocyte maturation in vitro and in vivo,

1989 ◽  
Vol 27 ◽  
pp. 39 ◽  
Author(s):  
P.M.M. Kastrop ◽  
M.M. Bevers ◽  
O.H.J. Destrée ◽  
Th.A.M. Kruip
Keyword(s):  
Protein Synthesis ◽  
Oocyte Maturation ◽  
Bovine Oocyte ◽  
Maturation In Vitro

Influence of vascular endothelial growth factor and Cysteamine on in vitro bovine oocyte maturation and subsequent embryo development

2015 ◽  
Vol 39 (10) ◽  
pp. 1090-1098 ◽  
Author(s):  
Juan Mateo Anchordoquy ◽  
Juan Patricio Anchordoquy ◽  
Juan Alberto Testa ◽  
Matías Ángel Sirini ◽  
Cecilia C. Furnus
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Vascular Endothelial ◽  
Bovine Oocyte ◽  
Endothelial Growth Factor

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

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