The effects of N-acetyl-L-cysteine supplementation on in vitro porcine oocyte maturation and subsequent fertilisation and embryonic development

2012 ◽  
Vol 24 (8) ◽  
pp. 1048 ◽  
Author(s):  
B. D. Whitaker ◽  
S. J. Casey ◽  
R. Taupier
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
The Other ◽  
Success Rates ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Intracellular Glutathione ◽  
Porcine Oocyte

The effects of supplementation with 1.5 mM n-acetyl-l-cysteine (NAC) during in vitro oocyte maturation were studied. Oocytes were supplemented with 1.5 mM NAC during maturation for 0 to 24 h, 24 to 48 h, or 0 to 48 h then subjected to IVF and embryo development. Oocytes were evaluated after maturation for intracellular glutathione concentration, superoxide dismutase and glutathione peroxidase activities and DNA fragmentation. Fertilisation and embryonic development success were also evaluated. There was no effect of treatment on intracellular glutathione concentrations, enzyme activities or fertilisation success rates. Supplementing NAC during maturation significantly decreased (P < 0.05) the percentage of oocytes with fragmented DNA compared with no NAC supplementation. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of oocytes with male pronuclei than for oocytes from the other treatment groups. There was no difference in the percentage of embryos cleaved by 48 h after IVF between treatment groups. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of embryos reaching the blastocyst stage by 144 h after IVF compared with the other treatment groups. These results indicate that supplementation of the oocyte maturation medium with 1.5 mM NAC, specifically during the last 24 h, improves male pronucleus formation and blastocyst development in pigs.

156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

2017 ◽  
Vol 29 (1) ◽  
pp. 187
Author(s):  
A. Goldacker ◽  
E. Winn ◽  
J. Z. Current ◽  
B. D. Whitaker
Keyword(s):  
Embryonic Development ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Success Rates ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Of Development ◽  
Fluid Supplementation ◽  
Chi Square Analysis

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P < 0.05) incidence of polyspermic penetrations and a significantly higher (P < 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P < 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P < 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
R. Krisher ◽  
A. Auer ◽  
K. Clark ◽  
K. Emsweller ◽  
S. Rogers ◽  
...  
Keyword(s):  
South Africa ◽  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Genetic Management ◽  
Blastocyst Development ◽  
Antidorcas Marsupialis

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P &lt; 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.

scholarly journals Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization

2020 ◽  
Vol 21 (10) ◽  
pp. 3692 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Sanghoon Lee ◽  
Soo-Hyun Park ◽  
Min Ju Kim ◽  
Hyo-Gu Kang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Cumulus Cell ◽  
Annexin V ◽  
Ros Generation ◽  
Vitro Fertilization ◽  
Porcine Oocyte ◽  
Mitochondrial Distribution

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 μM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 μM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

scholarly journals Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 355-366 ◽  
Author(s):  
Kazuhiro Kikuchi ◽  
Hans Ekwall ◽  
Paisan Tienthai ◽  
Yasuhiro Kawai ◽  
Junko Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Lipid Droplets ◽  
Early Embryonic Development ◽  
Oocyte Activation ◽  
Energy Status ◽  
Thin Sections ◽  
Porcine Oocyte

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.

185 PGI2 ANALOG INDUCES HIGH-QUALITY PORCINE BLASTOCYSTS IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 209 ◽  
Author(s):  
J.-S. Kim ◽  
G. Wee ◽  
B.-S. Song ◽  
J.-S. Park ◽  
X.-L. Jin ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mitogen Activated Protein Kinase ◽  
Blastocyst Formation ◽  
Prostaglandin I2 ◽  
Western Blots ◽  
Porcine Oocyte

Prostaglandin I2 (PGI2) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. In hatched embryos, prostaglandin I2 (PGI2) is related to implantation improvement, but its role during oocyte maturation and early embryo development remains controversial. Therefore, in this study, the effect of addition of a PGI2 analog during early porcine oocyte maturation on nuclear maturation, blastocyst formation, and pre-implantation embryonic quality was investigated. Porcine oocytes were matured in NCSU-23 medium supplemented with 10% (v/v) porcine follicular fluid, 10 ng mL-1 epidermal growth factor, 25 �M �-mercaptoethanol, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, 10 IU mL-1 hCG, and 1 �M PGI2 analog for 22 h, and then further cultured in maturation medium without PGI2 analog and hormones for 22 h. After fertilization in Tris-buffered (mTBM) medium for 6 h, presumptive porcine zygotes were cultured in the NCSU-23 medium supplemented with 4% BSA for 6 days. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). First, we confirmed that PGI2 analog-treated (90.0 � 2.6%) oocytes showed a higher proportion of the metaphase II stage than non-treated (65.7 � 1.4%) ones (P &lt; 0.05). Thus, to confirm the activities of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK), Western blots were performed in matured oocytes by using specific antibodies such as anti-cdc2 and anti-ERK1/2. The activities of MPF and MAPK were increased in porcine oocytes treated with PGI2 analog. In the PGI2 analog-treated group, polyspermic rate (17.9 � 13.3%) was reduced as compared with that of the non-treated group (35.8 � 9.4%). Furthermore, the rate (25.3%, 40/158) of blastocyst formation in the PGI2 analog-treated group was higher than in the non-treated group (19.7%, 27/137; P &lt; 0.05). Also, cell numbers of blastocysts were increased (29 � 2.5% vs. 39.6 � 1.4%) in the treated vs. the non-treated group. The numbers of fragmented DNA nuclei detected in the blastocyst stage by the TUNEL assay were decreased in the PGI2-treated group compared with the non-treated group (2.1% vs. 5.2%). In conclusion, direct roles of PGI2 during porcine oocyte maturation may involve reducing apoptosis and enhancing blastocyst quality.

scholarly journals Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence

2013 ◽  
Vol 25 (8) ◽  
pp. 1095 ◽  
Author(s):  
L. A. Frank ◽  
M. L. Sutton-McDowall ◽  
D. L. Russell ◽  
X. Wang ◽  
D. K. Feil ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Hexosamine Pathway ◽  
Hexosamine Biosynthesis ◽  
Necessary And Sufficient

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus–oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P < 0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P < 0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

scholarly journals Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Jason R Herrick ◽  
Amber M Brad ◽  
Rebecca L Krisher
Keyword(s):  
Embryonic Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Pentose Phosphate ◽  
The Other ◽  
Nuclear Maturation ◽  
Maturation In Vitro ◽  
Intracellular Content ◽  
Cytoplasmic Maturation

The objectives of this study were to manipulate metabolism of glucose through glycolysis and the pentose phosphate pathway (PPP) in porcine oocytes during in vitro maturation, and determine the effects of this manipulation on meiotic progression, intracellular glutathione (GSX) concentrations and embryonic development. Cumulus-oocyte complexes isolated from abattoir ovaries were matured (40–44 h) in Purdue Porcine Medium for maturation alone (control) or supplemented with pyrroline-5 carboxylate (PC, 0.1 μM; PPP stimulator), diphenyleneiodonium (DPI, 0.1 μM; PPP inhibitor), dinitrophenol (DNP, 10 μM; glycolytic stimulator), hexametaphosphate (HMP, 100 μM; glycolytic inhibitor), PC + HMP or DNP + DPI. At the conclusion of in vitro maturation, cumulus cells were removed and oocytes were randomly allocated for analysis of GSX, metabolism and nuclear maturation, or in vitro fertilization and embryo culture. Both DPI and DNP + DPI decreased (P ≤ 0.05) the activity of glycolysis and the PPP, increased (P ≤ 0.05) the percentage of immature oocytes, and decreased (P ≤ 0.05) the proportion of mature oocytes compared with control oocytes and oocytes from the other treatments. Embryonic development (cleavage and blastocyst stage) and the intracellular content of GSX were also decreased (P ≤ 0.05) following exposure to DPI or DNP + DPI compared with control oocytes and oocytes from the other treatments. Oocyte metabolism, nuclear maturation, GSX content and embryonic development were unaffected (P > 0.05) following exposure to PC, DNP, HMP or PC + HMP. Our results suggest that metabolism of glucose through the PPP and/or glycolysis plays a key role in the control of nuclear and cytoplasmic maturation of porcine oocytes in vitro.

scholarly journals Coenzyme Q10 Supplementation Effects on In Vitro Maturation, Fertilization, and Early Embryonic Development in Pigs

2019 ◽  
Vol 119 (2) ◽  
pp. 2
Author(s):  
Caitlin Streacker ◽  
Brian D. Whitaker
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Coenzyme Q10 ◽  
Early Embryonic Development ◽  
Mitochondrial Activity ◽  
Mitochondrial Membranes ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Development Rates

The objective of this study was to determine the reduction of polyspermic penetration, and increase of mitochondrial activity, in early pig embryonic development by supplementing different concentrations of coenzyme Q10 during oocyte maturation. Oocytes (n = 1,100) were supplemented during the last 24 h of maturation with 0 (control), 10, 50, or 100 μM of coenzyme Q10. After in vitro fertilization (IVF), embryos were evaluated for fertilization kinetics (penetration, polyspermic penetration, male pronuclear formation), and subsequent embryonic development and mitochondrial activity. Supplementation of 100 μM coenzyme Q10 was detrimental to the oocytes, as they had significantly lower (p < 0.05) fertilization kinetic and early embryonic development rates to the other treatment groups. There were no differences in fertilization kinetic and early embryonic development rates between the 0, 10 and 50 μM coenzyme Q10 treatment groups. Oocytes, matured in medium supplemented with 50 μM coenzyme Q10, ultimately developed into embryos with a significantly greater (p < 0.05) presence of intact mitochondrial membranes (observed at both 48 and 144 h post-IVF) compared to oocytes not supplemented with coenzyme Q10. In summary, supplementation of 100 μM coenzyme Q10 during oocyte maturation is detrimental, yet supplementation of 50 μM coenzyme Q10 leads to a higher occurrence of intact mitochondrial membranes in the in vitro produced pig embryos.

100 SEQUENTIAL VERSUS SINGLE-STEP MEDIUM FOR RHESUS MACAQUE EMBRYO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 180
Keyword(s):  
Embryonic Development ◽  
Embryo Culture ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Single Step ◽  
Blastocyst Development ◽  
Equal Variance ◽  
Significant Difference ◽  
Health Studies

The nonhuman primate (NHP) is a valuable translational model for human health studies and is widely used to investigate pre-implantation embryo development. Central to these investigations is the dependency on in vitro embryo culture (IVC). Since 2001, the single-step hamster embryo culture medium (HECM) has been the accepted standard for NHP IVC. With recent advances in formula optimization for IVC in human clinics, a re-examination of optimal NHP IVC media is warranted. Thus, two types of commercially available IVC media routinely used in human applications were compared with HECM-9: Global (single-step; LifeGlobal Group, Guilford, CT, USA), and Quinns Advantage (sequential; SAGE, Trumbull, CT, USA). Normally cycling, adult rhesus monkeys (n = 3) underwent controlled ovarian stimulations, and follicles were aspirated via laparoscope. Recovered ova were fertilized in vitro and the resultant zygotes (n = 138) were cultured for 9 days in HECM-9, Global, or Quinns with 10% protein supplement at 37.5°C in humidified tri-gas (6% CO2, 5% O2, and 89% N). Single-step media (HECM-9 and Global) were refreshed every two days while embryos were cultured for Days 1–3 in Quinns Advantage Cleavage medium without being replaced and in Quinns Advantage Blastocyst medium for Days 4–9 with medium changes every 2 days. Embryos were observed for cleavage, compaction, and blastocyst development. Proportional data with equal variance and normal distribution were analysed by one-way ANOVA, and significance was determined post-hoc by the Holm-Sidak method with P < 0.05. Developmental stage data ± s.e.M are presented in Table 1; a change in superscript indicates a significant difference within the column. There was no difference in embryonic cleavage or morula compaction between the three culture media evaluated, indicating no obvious differences in their effects on embryonic development 1 to 3 days after fertilization. However, a greater proportion of blastocysts developed in Global medium compared with HECM-9, and though it was not statistically different, embryos cultured in Global tended to reach the blastocyst stage more frequently than those in Quinns. Although not significant due to large variances in each group, blastocyst expansion also tended to occur more frequently in Global medium than in HECM-9 or Quinns. Taken together, these data indicate that single-step Global is as supportive of early embryonic development as HECM-9 but is better formulated to facilitate later stage differentiation and would be better suited for use in updated standard NHP IVC protocols. Table 1.Cleavage, compaction, blastocysts, and expansion of embryos in HECM-9, Global, and Quinns media

241 STRATEGIES TO IMPROVE GLUTATHIONE CONTENT OF IN VITRO-MATURED BOVINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 200 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Colorimetric Assay ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Experimental Conditions ◽  
Cell Counts

Glutathione is the main non-enzymatic defense against oxidative stress and a critical part of oocyte maturation and normal fertilization. Our aim was to test different strategies to manipulate cellular glutathione (GSH) content of bovine in-vitro-matured (IVM) oocytes and study the development of embryos produced from such oocytes. The reducing agents lipoic acid (LA, intracellular) and dihydrolipoic acid (DHLA, extracellular) were compared to the cell-permeable reduced glutathione (GSH) donor glutathione ethyl ester (OET) for their effect on oocyte GSH content, oocyte maturation, and blastocyst development (OET only). Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured for 24 h in a humidified atmosphere of 6% CO2 at 38.5�C in modified tissue culture medium (mTCM199) supplemented with 2% (LA, DHLA) or 10% (OET) fetal calf serum (FCS; Gibco, Grand Island, NY, USA), 0.1 IU bLH and 0.1 IU bFSH (Sioux Biochemicals, Sioux City, IA, USA). COCs were matured in the presence of either LA (100 µm) or DHLA (100 µm) alone or in combination with L-cystine (CYS; 0.6 mm), CYS alone, or OET at 1, 3, and 5 mm. COCs matured under control and experimental conditions were denuded of cumulus cells (40 IU hyaluronidase) and scored for maturity. GSH content of MII oocytes was determined by colorimetric assay (Northwest Life Science Specialties, LLC, Vancouver, WA, USA). Oocytes matured in OET were inseminated with frozen/thawed bull sperm (2 � 106 mL-1), cultured to the blastocyst stage (COOK bovine medium, COOK Australia, Brisbane, Queensland, Australia), and subjected to differential cell count (propidium iodide/Hoechst). GSH levels (mean � SEM) and developmental data (percentage) are expressed for n = 18–73 oocytes or embryos and were analyzed by ANOVA or chi-square test (significance, P ≤ 0.05). LA alone failed to increase oocyte GSH content over 2% FCS control levels (6.98 � 0.22 pmol/oocyte v. 5.26 � 0.4 pmol/oocyte). DHLA alone significantly increased oocyte GSH content (9.64 � 0.8 pmol/oocyte) compared to both LA and controls (10% FCS; 4.78 � 0.36 pmol/oocyte). CYS alone (10.18 � 0.58 pmol/oocyte) or in combination with LA (10.84 � 0.37 pmol/oocyte) or DHLA (9.75 � 0.66 pmol/oocyte) significantly increased GSH compared to controls. GSH content of MII oocytes matured in 5 mm OET (8.35 � 0.35 pmol/oocyte) was significantly higher compared to control (5.07 � 0.32 pmol/oocyte), 1 mm (4.21 � 0.18 pmol/oocyte), and 3 mm (7.12 � 0.35 pmol/oocyte) OET treatments. Maturation rates of oocytes were significantly reduced in 2% FCS (51.1–72%) compared to 10% FCS (90.5%). OET treatment (1–5 mm) did not significantly alter maturation rate compared to control (75–89.8%). Blastocyst development of IVM oocytes treated with 1 mm OET (22.5%) was significantly lower compared to 3 mm (42.3%) and 5 mm (41.1%) OET but not to control (33.6%). Blastocysts from IVM oocytes treated with 5 mm OET had significantly higher cell counts compared to controls (126 � 6.4 cells v. 100.8 � 5.2 cells). Bovine IVM is a valuable model for testing the efficacy of various strategies to increase oocyte cellular GSH. Both strategies improve oocyte GSH levels, and an increase in blastocyst cell number occurred with GSH donor treatment (5 mm OET).

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