45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

Effects of metformin on fertilisation of bovine oocytes and early embryo development: possible involvement of AMPK3-mediated TSC2 activation

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 58-67 ◽  
Author(s):  
Olympia Pikiou ◽  
Anna Vasilaki ◽  
George Leondaritis ◽  
Nikos Vamvakopoulos ◽  
Ioannis E. Messinis
Keyword(s):  
Embryo Development ◽  
Adenosine Monophosphate ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Downstream Target ◽  
Protein Levels ◽  
Negative Effect ◽  
Bovine Oocytes ◽  
Dose Dependent

SummaryStudies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 μM) on the percentage of cultured cumulus–oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥8-cell embryos (1 μM, P < 0.05; 10 μM, P < 0.01; and 0.1 μM, 10 μM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 μM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 μM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.

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.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

Effects of EPA on bovine oocytes matured in vitro with antioxidants: Impact on the lipid content of oocytes and early embryo development

2020 ◽  
Vol 146 ◽  
pp. 152-161 ◽  
Author(s):  
Noelia Nikoloff ◽  
Anabella Campagna ◽  
Carolina Luchetti ◽  
Ana C. Carranza-Martín ◽  
Ana M. Pascua ◽  
...  
Keyword(s):  
Embryo Development ◽  
Lipid Content ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Bovine Oocytes

scholarly journals Exosomes as a Potential Tool for Supporting Canine Oocyte Development

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1971
Author(s):  
Seok Hee Lee ◽  
Islam M. Saadeldin
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Biological Function ◽  
Cumulus Cells ◽  
Reproductive Organ ◽  
Early Embryo ◽  
Molecular Signaling ◽  
Early Embryo Development ◽  
Potential Tool

The canine oviduct is a unique reproductive organ where the ovulated immature oocytes complete their maturation, while the other mammals ovulate matured gametes. Due to their peculiar reproductive characteristics, the in vitro maturation of dog oocytes is still not wellestablished compared with other mammals. Investigations of the microenvironment conditions in the oviductal canal are required to establish a reliable in vitro maturation system in the dog. Previous studies have suggested that the oviduct and its derivatives play a key role in improving fertilization as well as embryo development. In particular, the biological function of oviduct-derived exosomes on sperm and early embryo development has been investigated in porcine, bovine, and murine species. However, the information about their functions on canine cumulus-oocyte complexes is still elusive. Recent canine reproductive studies demonstrated how oviduct-derived extracellular vesicles such as microvesicles and exosomes interact with oocyte-cumulus complexes and how they can play roles in regulating canine cumulus/oocyte communications. In this review, we summarize the physiological characteristics of canine oviduct-derived exosomes and their potential effects on cumulus cells development as well as oocyte in vitro maturation via molecular signaling pathways.

254 EFFECTS OF OXYGEN TENSION AND OOCYTE DENSITY DURING IN VITRO MATURATION ON THE LEVELS OF REACTIVE OXYGEN SPECIES IN BOVINE OOCYTES AND MATURATION MEDIUM

2013 ◽  
Vol 25 (1) ◽  
pp. 274
Author(s):  
A. B. Giotto ◽  
A. C. G. Guimarães ◽  
C. G. M. Gonçalves ◽  
N. P. Folchini ◽  
C. I. I. U. F. Machado ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxygen Tension ◽  
In Vitro Maturation ◽  
Early Embryo ◽  
Ros Production ◽  
Oxygen Species ◽  
Early Embryo Development ◽  
Treatment Groups ◽  
Bovine Oocytes

The reactive oxygen species (ROS) produced by animal cells and at physiological levels are responsible for several cellular functions. However, when there is an imbalance between ROS production and the antioxidant system in the cell, oxidative stress occurs and causes severe cell damage. In oocytes, ROS can affect the dynamics of maturation and early embryo development processes. Oxygen tension and the density of oocytes by medium volume during in vitro maturation (IVM) can influence ROS production. The aim of this study was to evaluate the influence of the association between oxygen tension (5 or 20%) and different oocyte densities during IVM (1 : 10 or 1 : 20 oocytes µL–1 of medium) on the ROS levels in oocytes and medium. Bovine oocytes (n = 420) were obtained from slaughterhouse ovaries by aspiration of 2- to 8-mm follicles. Quality I and II oocytes (De Loss et al. 1989 Gamete Res. 24, 197–204) were homogeneously distributed into groups of 15 oocytes per treatment: Treatment (T) 1 = 1 : 10 in 5% of O2; T2 = 1 : 10 in 20% of O2; T3 = 1 : 20 in 5% of O2; and T4 = 1 : 20 in 20% of O2. The oocytes were matured in TCM-199 supplemented with 10% oestrous mare serum, 100 µg mL–1 of epidermal growth factor, 50 µg mL–1 of LH, 5 µg mL–1 of FSH, and 22 µg mL–1 of pyruvate for 22 to 24 h at 39°C, in 5% CO2 and saturated humidity. To assay ROS production, denuded oocytes and 60-µL samples of IVM medium were evaluated by the spectrofluorometric method with 2′7′-dichlorofluorescein-diacetate, in which the fluorescence intensity emission was considered an indicator of ROS production and measured by a spectrofluorophotometer. The ROS production in oocytes and in IVM medium was expressed as units of fluorescence (UF); data were analysed by ANOVA and Duncan’s test with a 5% level of significance. Seven replications were performed. In treatment groups T1 and T3, the ROS production in oocytes was higher (P < 0.05) than in oocytes of treatment groups T2 and T4 (13.53 and 18.78 UF v. 7.92 and 6.15 UF, respectively). The ROS production in IVM medium was higher in the T1 (23.86 UF) and T2 (24.12 UF) treatment groups than in the T3 (18.78 UF) and T4 (18.57 UF) treatment groups. These results suggest an increase in ROS production in IVM oocytes under a 5% O2 atmosphere in relation to a 20% O2 atmosphere, irrespective of the oocyte density by volume of IVM medium. On the other hand, the accumulation of ROS in IVM medium seemed higher when the oocyte density was 1 oocyte to 10 µL of IVM medium, independent of the oxygen tension used. A higher level of ROS in 5% O2 tension may be caused by competition for O2 between oocyte and cumulus cells, causing a reduction in O2 levels and changing the availability of O2 to energy generation in oocytes and consequently increasing ROS generation. In this respect, 5% O2 during IVM may contribute to the onset of oxidative stress in oocytes, which may compromise fertilization and early embryo development. Further research is necessary to clarify esterase activity in oocytes and the addition of exogenous peroxidase to validate the assay. Financial support: FAPERGS (1011575) and CNPq (501763/2009).

scholarly journals Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro

Reproduction ◽  
2001 ◽  
pp. 155-163 ◽  
Author(s):  
QY Sun ◽  
GM Wu ◽  
L Lai ◽  
KW Park ◽  
R Cabot ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Mitochondrial Translocation ◽  
Strong Staining ◽  
Development In Vitro ◽  
Pig Oocyte

The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

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