250 EFFECT OF LOW OXYGEN TENSION ATMOSPHERE AND MATURATION MEDIA ON IN VITRO-MATURED SWINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 223
Author(s):  
M. G. Marques ◽  
F. R. O. de Barros ◽  
M. D. Goissis ◽  
P. V. Cavalcanti ◽  
A. C. Nicacio ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Cortical Granule ◽  
Low Oxygen ◽  
Cortical Granules ◽  
Significance Level ◽  
Migration Rates ◽  
Nuclear Maturation ◽  
Low Oxygen Tension ◽  
Maturation Rate

The aim of this study was to evaluate the efficiency of a low oxygen tension atmosphere (5% CO2, 5% O2, and 90% N2) on swine oocyte maturation in chemically defined media or when supplemented with porcine follicular fluid (PFF). Briefly, oocytes were in vitro matured for 44 h in TCM-199 with 10% PFF or 0.1% PVA added, under a low oxygen tension atmosphere or a normal oxygen tension atmosphere (5% CO2 in air, approximate 20% O2). At 0 and 44 h of maturation, cumulus oophorus cells were removed. To evaluate the migration of cortical granules, oocytes were fixed, permeabilized, and incubated in 100 μg of FITC-PNA mL–1 for 30 min. Oocytes were then incubated in 10 μg mL–1 of Rnase for 30 min and in 10 μg mL–1 of propidium iodide for 10 min to verify nuclear maturation by confocal microscopy (Zeiss LSM 510 Meta). Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify oxidative stress. Data were analyzed by the SAS System for Windows (2000). The nonparametric ANOVA NPAR1WAY procedure was applied to evaluate nuclear maturation rate by comparing groups in pairs. Migration of cortical granules and HSP70 content were analyzed using PROC GLM (LSD test of means). The effects of treatment and manipulation were verified in all analyses. The significance level was 5%, and data were presented as means ± SEM. Results indicated that the percentage of metaphase II oocytes did not differ among groups after 44 h of maturation [PFF 5% O2 (89.16 ± 3.73a), PFF 20% O2 (86.59 ± 6a), PVA 5% O2 (79.62 ± 8.22a), and PVA 20% O2 (93 ± 5.17a)]. However, these groups were different from the 0-h group (0 ± 0b). Results for the percentage of cortical granule migration showed that 0-h oocytes (38.92 ± 2.75a) had lower migration rates compared with other groups. After 44 h of maturation, migration of the cortical granule rate of the PFF-supplemented group under a 5% O2 atmosphere (61.66 ± 3.21b) was different when compared with the PVA 20% O2 group (50.97 ± 3.48c). The other groups showed intermediate results, but without statistical differences [PFF 20% O2 (58.51 ± 2.5bc) and PVA 5% O2 (53.75 ± 3.14bc)] for the migration of cortical granules. Moreover, no difference at pixel quantification of HSP70 was observed among groups after 44 h of maturation [PFF 5% O2 (116.45 ± 40.94a), PFF 20% O2 (44.44 ± 12.66a), PVA 5% O2 (29.95 ± 7.95a), and PVA 20% O2 (58.49 ± 22.2a)], although these groups were different from the 0-h group (247.41 ± 38.59b). Although the HSP content decreased throughout in vitro maturation of swine oocytes under the low and high oxygen tension atmospheres, it can be concluded that a low oxygen tension atmosphere did not affect nuclear maturation and rates of cortical granule migration regardless of maturation media supplementation. Financial support: FAPESP (grant no. 05/01420-7).

247 EFFECTS OF CYSTEINE IN IVM MEDIA ON IN VITRO MATURATION UNDER LOW OXYGEN TENSION, IN VITRO FERTILIZATION, AND IN VITRO CULTURE OF PORCINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 203
Author(s):  
N. V. Linh ◽  
D. N. Q. Thanh ◽  
M. Ozawa ◽  
B. X. Nguyen ◽  
K. Kikuchi ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Blastocyst Stage ◽  
Low Oxygen ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Low Oxygen Tension ◽  
High O2 ◽  
Group 1

Cysteine is considered to promote male pronuclear (MPN) formation in porcine through oocyte glutathione (GSH) synthesis (Yoshida et al. 1993 Biol. Reprod. 49, 89–94). The GSH has an important role in providing cells with a redox state and in acting to protect cells from toxic effects of oxidative damage (Meister et al. 1976 AM Rev. Biochem. 45, 559–604). However, such previous investigations were carried out under high O2 tension (20% O2) incubation conditions. Here we simply study IVM-IVF-IVC competence of porcine oocytes matured in IVM media supplemented with cysteine of different concentrations under low oxygen tension (5% O2). Cumulus–oocyte complexes (COCs) from prepubertal gilts were collected, matured, and fertilized in vitro according to Kikuchi et al. (2000 Biol. Reprod. 66, 1033–1041). COCs were cultured in IVM medium supplemented with 0 (Group 1; control), 0.05 (Group 2), 0.1 (Group 3), 0.2 (Group 4), and 0.6 mm (Group 5) cysteine under low oxygen tension. Nuclear maturation of oocytes, fertilization status, and number of cells in resultant embryos were assessed with orcein staining; also, the GSH content of IVM oocytes was measured by the method described by Ozawa et al. (2002 Reproduction 124, 683–689). Maturation rates of Groups 1–5 were 68.2 � 3.2, 70.6 � 7.7, 69.7 � 15.9, 75.9 � 7.7, and 68.8 � 8.0%, respectively, indicating no difference in maturation competence among the groups (P > 0.05 by ANOVA). The rates of sperm penetration, MPN formation (95.9 � 2.4, 100 � 0, 92.8 � 4.7, 94.0 � 4.1, and 92.4 � 2.7%, respectively), monospermy, and even blastocyst rates after 6 days of IVC were not different among the groups (P > 0.05 by ANOVA). Moreover, the cell numbers of blastomeres in blastocysts (38.68 � 3.5, 40.1 � 3.1, 37.5 � 3.0, 36.2 � 3.3, and 43.8 � 4.0, respectively) were uniformly the same among the groups (P > 0.05 by ANOVA). However, GSH content of IVM oocytes increased significantly (P < 0.05 by ANOVA) as the concentration of cysteine increased (12.2 � 0.6, 14 � 0.8, 15.1 � 0.5, 16.4 � 0.4, and 16.4 � 0.5 pmol/oocyte, respectively). The GSH level of oocytes in Group 1 (control) seems to be higher than that reported by Aberydeera et al. (1998 Biol. Reprod. 58, 213–218), who matured porcine oocytes under high O2 tension. This may reflect the effect of low O2 tension and explain the same developmental rate to the blastocyst stage as that of oocytes matured in the media supplemented with cysteine in this study. In conclusion, an addition of 0.05–0.6 mm cysteine during IVM, under 5% O2 tension, of porcine oocytes significantly increased intracellular GSH synthesis according to its concentration. However, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation, and subsequent embryonic development to the blastocyst stage. Thus, O2 tension during IVM of oocytes is suggested to be important for the in vitro production of porcine blastocysts.

Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation

Zygote ◽  
2021 ◽  
pp. 1-11
Author(s):  
Nasser Ghanem ◽  
Dalia Abd-El Rahman Ahmed ◽  
Sherif Mohamed Dessouki ◽  
Marwa Said Faheem ◽  
Ahmed Yehia Gad ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
High Oxygen ◽  
Mitochondrial Activity ◽  
Low Oxygen ◽  
Cortical Granules ◽  
Molecular Changes ◽  
Oxygen Levels ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Low Oxygen Tension

Summary This study was conducted to monitor the cellular and molecular changes of buffalo cumulus–oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB− and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB− group. BCB− oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB− under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB−) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB− oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.

Embryo culture at a reduced oxygen concentration of 5%: a mini review

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 355-361 ◽  
Author(s):  
R. Sciorio ◽  
G.D. Smith
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Atmospheric Oxygen ◽  
Embryo Implantation ◽  
Critical Role ◽  
Low Oxygen ◽  
Physiological Level ◽  
Low Oxygen Tension

SummaryThe optimum oxygen tension for culturing mammalian embryos has been widely debated by the scientific community. While several laboratories have moved to using 5% as the value for oxygen tension, the majority of modern in vitro fertilization (IVF) laboratory programmes still use 20%. Several in vivo studies have shown the oxygen tension measured in the oviduct of mammals fluctuates between 2% and 8% and in cows and primates this values drops to <2% in the uterine milieu. In human IVF, a non-physiological level of 20% oxygen has been used in the past. However, several studies have shown that atmospheric oxygen introduces adverse effects to embryo development, not limited to numerous molecular and cellular physiology events. In addition, low oxygen tension plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development to the blastocyst stage. Collectively, this improves embryo implantation potential. However, clinical studies have yielded contradictory results. In almost all reports, some level of improvement has been identified in embryo development or implantation, without any observed drawbacks. This review article will examine the recent literature and discusses ongoing efforts to understand the benefits that low oxygen tension can bring to mammal embryo development in vitro.

scholarly journals Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277 ◽  
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Oxygen Tension ◽  
Polar Body ◽  
Ros Production ◽  
Low Oxygen ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Cytoplasmic Maturation ◽  
Bovine Oocytes

<p>Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the <em>in vitro </em>maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P&gt;0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P&lt;0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P&lt;0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P&lt;0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P&lt;0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of <em>in vitro </em>fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).</p>

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