110 VITRIFICATION DEVICES AFFECT DEVELOPMENTAL COMPETENCE AND BIOCHEMICAL PROPERTIES OF IVM OVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 163 ◽  
Author(s):  
S. Succu ◽  
G. G. Leoni ◽  
F. Berlinguer ◽  
F. Mossa ◽  
M. Galioto ◽  
...  
Keyword(s):  
Standard Condition ◽  
Biochemical Properties ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Parthenogenetic Activation ◽  
Molecular Alterations ◽  
Mapk Activity

This study was designed to evaluate the effects of 3 different vitrification devices on the developmental ability and on the maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of ovine oocytes in vitro-matured. Cumulus-oocytes complex (COCs) were in vitro-matured in TCM-199 supplemented with 10% FCS, 10 �L/mL of FSH/LH, and 1 �g/mL estradiol at 39�C and 5% CO2 atmosphere. For vitrification, oocytes were incubated in HEPES-buffered TCM-199 containing 20% FCS supplemented with DMSO (7.5%) and ethylene glycol (7.5%). After 3 min, oocytes were loaded into the same medium containing 0.5 m sucrose, 16.5% DMSO, and 16.5% EG, and then immersed into N2 using open pull straw (OPS; Vajta et al. 1998 Mol. Reprod. Dev.), cryoloop (CL; Lane et al. 1999 Nat. Biotechnol.), or cryotop (CT; Kuwayama and Kato 2000 J. Assist. Reprod. Genet.). After warming a part of oocytes were fertilized and cultured in vitro up to blastocyst stage in standard conditions (Leoni et al. 2005 Anim. Reprod. Sci., in press). The fertilization (23.8%, 31.6%, and 36.8% vs. 61.5%) and blastocyst rates (0, 12.5, and 0% vs. 50.0%) were lower for oocytes vitrified in OPS, CL, and CT, compared with control group. Control and vitrified IVM oocytes were valued for MPF and MAPK activity at 0 and 2 h after warming. In both post-warming experimental groups, the MAPK activity did not differ from control group. Immediately post-warming, MPF activity was lower in the vitrified groups compared with control oocytes (P < 0.01). If 100% is assigned to MPF activity in the control oocytes, those in the OPS, CL, and CT groups were, respectively, 63.3%, 65.5%, and 26.2%. After warming and culture for 2 h in standard condition, the activity of MPF was restored to values similar to control oocytes (87.0 and 76.8%, respectively) in the OPS and CL groups, whereas it was at the similar value in the CT group. To evaluate if the lowered MPF activity could cause parthenogenetic activation, the vitrified-warmed oocytes were cultured in SOF + 2% oestrus sheep serum in 5% O2, 5% CO2; after 27 h of culture, the oocytes were fixed and stained with propidium iodide to evaluate chromatin configuration. Results showed significantly higher parthenogenetic activation rates in the CT group compared with OPS and CL groups (54.5% vs. 22.6 and 27.4%, respectively). Our results indicate that the success of cryopreservation of the ovine oocyte is still very limited. The use of different vitrification devices not only modifies the ability to survive cryopreservation and developmental competence of oocytes but is also associated with important molecular alterations in the warmed oocyte cytoplasm. This work was supported by Cofin 2003.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

Histone deacetylase inhibitor trichostatin A affects porcine oocyte maturation in vitro

2014 ◽  
Vol 26 (6) ◽  
pp. 806 ◽  
Author(s):  
Yong-Xun Jin ◽  
Ming-Hui Zhao ◽  
Zhong Zheng ◽  
Jung-Suk Kwon ◽  
Seul-Ki Lee ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Germinal Vesicle ◽  
Oocyte Quality ◽  
Histone Deacetylation ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Germinal Vesicle Breakdown ◽  
Porcine Oocyte

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24 h and then continuously cultured for another 24 h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 36-45
Author(s):  
Jaqueline Sudiman ◽  
Alice Lee ◽  
Kheng Ling Ong ◽  
Wu Zi Yuan ◽  
Sarah Jansen ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cortical Granule ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Different Response

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

scholarly journals Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 741
Author(s):  
Dongjin Oh ◽  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Junchul-David Yoon ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Interleukin 7

Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

scholarly journals 101 OVARIAN RESPONSE AND DEVELOPMENTAL COMPETENCE OF OOCYTES COLLECTED BY OPU IN SHEEP TREATED WITH GnRH ANTAGONIST

2005 ◽  
Vol 17 (2) ◽  
pp. 201
Author(s):  
F. Berlinguer ◽  
A. Gonzalez-Bulnes ◽  
S. Succu ◽  
G. Leoni ◽  
I. Rosati ◽  
...  
Keyword(s):  
Single Dose ◽  
Gnrh Antagonist ◽  
Ovarian Response ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Gnrh Antagonists ◽  
Group A ◽  
Group B

The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were collected by OPU technique. Follicular growth was monitored by transrectal ultrasonography from Day 7 to Day 11. Collected oocytes were matured, fertilized, and cultured in vitro up to blastocyst stage under standard conditions used in our laboratory (Berlinguer F et al. 2004 Theriogenology 61, 1477–1486). After IVF, uncleaved oocytes were stained with acetolacmoid to evaluate chromatin configuration, while the cleaved ones were cultured in SOF + 0.4% BSA up to the blastocyst stage. Data were analyzed by ANOVA statistical analysis after arcsine transformation of the value percentages. Ultrasonographic monitoring showed a significant increase in the number of follicles (mean ± SEM) present in the ovaries from Day 8 to Day 11 of treatment in group A compared to group B (Day 8: 19 ± 5.1 vs. 13 ± 3.4, P > 0.05; Day 9: 20.1 ± 4.6 vs. 14.1 ± 2.4, P > 0.001; Day 10: 22.5 ± 6.1 vs. 14.7 ± 2.7, P > 0.001; Day 11: 25.3 ± 5.1 vs. 20.5 ± 4.1, P > 0.05), thus confirming that GnRH antagonist administration enhances ovarian response to exogenous FSH stimulation. On the other hand, oocytes collected from untreated sheep lead to a higher blastocyst output (P = 0.014), as illustrated in the table. These results indicated that although GnRH antagonist administration caused a significant increase in the ovarian response to the hormonal treatment, the final blastocyst output was significantly lower compared to that of the control group. This finding seems to suggest an impairment in the developmental competence of treated sheep oocytes. Table 1. In vitro maturation, fertilization, and developmental capacity of oocytes collected from follicles of GnRH antagonist-treated (group A) and untreated (group B) sheep This work was supported by funds from the Spanish MEC (projects SC 00-051-C3.1 and HI2002-0004) and the Italian MIUR (cofin).

172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 194
Author(s):  
M. Ozawa ◽  
T. Nagai ◽  
M. Fahrudin ◽  
N. W. K. Karja ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Redox Status ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Normal Embryo ◽  
Control Group ◽  
Intracellular Redox Status ◽  
Intracellular Redox

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H2O2 level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H2O2. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 �M GSH (29.6 � 2.7% or 30.4 � 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 � 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 � 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 � 2.1 to 49.2 � 2.1) and lowered ratios of apoptotic cells (6.2 � 0.6% to 7.0 � 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 � 2.0, apoptosis rate = 11.1 � 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.

116 TAUROURSODEOXYCHOLIC ACID PREVENTS ENDOPLASMIC RETICULUM STRESS-INDUCED APOPTOSIS IN PREIMPLANTATION PIG EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 158
Author(s):  
J.-S. Kim ◽  
K.-S. Lee ◽  
B.-S. Song ◽  
J.-Y. Zhang ◽  
Y.-K. Choo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Tauroursodeoxycholic Acid ◽  
Preimplantation Stage ◽  
Induced Apoptosis

Apoptosis is an important determinant for the normal development of preimplantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. The efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting classic pathways of apoptosis. Therefore, we explored the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis in preimplantation porcine embryos. Also, TM (tunicamycin; an ER stress inducing chemical reagent) was used to investigate the effect of ER-stress on pig embryo development. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 μ g mL–1 TUDCA or 1 μg mL–1 (TM) for 6 days at 39°C, 5% CO2 in air. All data were analyzed by using Duncan test of ANOVA by Statistical Analysis System (SAS). When treated with TM during culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage compared with 27.4% (28/102) of the embryos in the control group (P < 0.05). We also confirmed that TM stimulates up-regulation of ER stress response genes, such as XBP-1 mRNA, and induces a high rate of apoptosis. Whereas the frequency of blastocyst formation in the TUDCA-treated group was increased compared with that in the control group (32.8%, 49/149 v. 22.2%, 32/144), P < 0.05). Furthermore, the blastocyst cell number was enhanced (30.6 v. 39.5) and apoptosis reduced (TUNEL positive nuclei number, 6.0 v. 3.2) by TUDCA treatment in pig embryos. As the result of real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-xl gene was increased in the blastocyst stage by TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also confirmed that TUDCA decreases the rate of TM-induced apoptosis in preimplantation stage pig embryos. Our results indicate that TUDCA improves the developmental competence of porcine embryos by modulating the ER stress-induced apoptosis during preimplantation stage.

scholarly journals 3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1101
Author(s):  
Daniela Bebbere ◽  
Stefano Mario Nieddu ◽  
Federica Ariu ◽  
Davide Piras ◽  
Sergio Ledda
Keyword(s):  
Mammalian Species ◽  
3D Culture ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Beneficial Effects ◽  
Fumed Silica Nanoparticles ◽  
Liquid Marble

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus–oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

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