Supplementation with asiatic acid during in vitro maturation improves porcine oocyte developmental competence by regulating oxidative stress

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

Download Full-text

α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes

Reproduction Fertility and Development ◽

10.1071/rd17525 ◽

2018 ◽

Vol 30 (12) ◽

pp. 1728 ◽

Cited By ~ 3

Author(s):

M. Arias-Álvarez ◽

R. M. García-García ◽

J. López-Tello ◽

P. G. Rebollar ◽

A. Gutiérrez-Adán ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Rabbit Model ◽

Developmental Competence ◽

Antioxidant Agents ◽

Junction Protein

The developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400 µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus–oocyte complexes (COCs) after IVM with 100 μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

Download Full-text

327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab327 ◽

2006 ◽

Vol 18 (2) ◽

pp. 271 ◽

Cited By ~ 1

Author(s):

T. S. Hussein ◽

R. B. Gilchrist ◽

J. G. Thompson

Keyword(s):

In Vitro Maturation ◽

Cumulus Cell ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Oocyte ◽

Paracrine Factors ◽

Cell Functions ◽

Oocyte Developmental Competence ◽

Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

Download Full-text

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab205 ◽

2010 ◽

Vol 22 (1) ◽

pp. 260

Author(s):

M. Bertoldo ◽

P. K. Holyoake ◽

G. Evans ◽

C. G. Grupen

Keyword(s):

Cell Morphology ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Developmental Competence ◽

Oocyte Developmental Competence ◽

Before And After ◽

Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

Download Full-text

342 DISTINCT EFFECTS OF FOLLICULAR FLUID ON CUMULUS CELL APOPTOSIS AND PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab342 ◽

2007 ◽

Vol 19 (1) ◽

pp. 286

Author(s):

C. G. Grupen ◽

T. S. Hussein ◽

S. J. Schulz ◽

D. T. Armstrong

Keyword(s):

Oxidative Stress ◽

Follicular Fluid ◽

Cell Apoptosis ◽

Polar Body ◽

Cumulus Cell ◽

Developmental Competence ◽

Potent Inducer ◽

Oocyte Developmental Competence ◽

Cytoplasmic Maturation

Supplementing medium with follicular fluid (FF) during in vitro maturation (IVM) enhances the developmental competence of porcine oocytes, indicating that factors present in FF are beneficial to cytoplasmic maturation. Previous findings suggest that porcine FF contains high levels of superoxide dismutase activity and exerts a beneficial effect on cytoplasmic maturation by protecting oocytes from oxidative stress (Tatemoto et al. 2004 Biol. Reprod. 71, 1150–1157). Since oxidative stress is a potent inducer of apoptosis, the aim of the present study was to examine the temporal effects of FF during IVM on cumulus cell apoptosis and oocyte developmental competence. Ovaries of prepubertal pigs were collected from a local abattoir and antral follicles, 3 to 7 mm in diameter, were aspirated. Cumulus–oocyte complexes (COCs) with at least 3 uniform layers of compact cumulus cells (CCs) were recovered, washed, and transferred to maturation medium (MM) with or without 25% FF. At 22 h of IVM, COCs from each group were washed and transferred to fresh MM with or without 25% FF, forming 4 groups: -FF/-FF, -FF/+FF, +FF/-FF, and +FF/+FF. Cohorts of COCs were TUNEL stained at 22 and 44 h of IVM using the In Situ Cell Death Detection kit (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer's instructions, and apoptotic CCs were visualized using confocal microscopy. Oocytes denuded at 44 h, that had a polar body, were treated with ionomycin and 6-dimethylaminopurine to induce parthenogenetic development, and were cultured for 7 days in NCSU-23 medium at 38.5°C in 5&percnt; O2, 5&percnt; CO2, and 90&percnt; N2. Data were subjected to ANOVA and Tukey's post-hoc test. At 22 h of IVM, the presence of FF reduced the proportion of apoptotic CCs in COCs (2.1&percnt; vs. 4.6&percnt;). COCs matured with FF from 22 to 44 h of IVM had much lower proportions of apoptotic CCs (+FF/+FF: 0.9&percnt;; −FF/+FF: 2.6&percnt;) compared with those matured without FF (+FF/−FF: 10.3&percnt;; −FF/−FF: 17.8&percnt;). The rate of maturation to the metaphase-II stage was greater when oocytes were matured with FF from 0 to 22 h of IVM (−FF/−FF: 68.6&percnt;; −FF/+FF: 72.8&percnt;; +FF/−FF: 89.2&percnt;; +FF/+FF: 86.2&percnt;). Maturation without FF for the entire IVM interval reduced the proportion of activated oocytes that formed blastocysts compared with the other groups (−FF/−FF: 25.1&percnt;; −FF/+FF: 44.6&percnt;; +FF/−FF: 46.6&percnt;; +FF/+FF: 47.3&percnt;). Despite a 4-fold difference in the proportion of apoptotic CCs between COCs of the +FF/−FF and −FF/+FF groups, exposure to FF for the first or second half of IVM was as beneficial to oocyte developmental competence as exposure to FF for the entire IVM interval. This suggests that the protective effect of FF in reducing oxidative stress on oocytes during IVM is distinct from the effect on oocyte developmental competence.

Download Full-text

Quercetin improves developmental competence of mouse oocytes by reducing oxidative stress during in vitro maturation

Annals of Animal Science ◽

10.1515/aoas-2017-0029 ◽

2018 ◽

Vol 18 (1) ◽

pp. 87-98

Author(s):

Seyede Zahra Banihosseini ◽

Marefat Ghaffari Novin ◽

Hamid Nazarian ◽

Abbas Piryaei ◽

Siavash Parvardeh ◽

...

Keyword(s):

Oxidative Stress ◽

Embryo Development ◽

In Vitro Maturation ◽

Mature Oocyte ◽

Developmental Competence ◽

Nuclear Maturation ◽

Mouse Oocytes ◽

Blastocyst Rate ◽

And Control

Abstract Quercetin is a natural flavonoid with strong antioxidant activity. In the present study, we evaluate the influence of different concentrations of quercetin (QT) on intracytoplasmic oxidative stress and glutathione (GSH) concentration, during in vitro maturation (IVM) and fertilization in mouse oocytes. IVM was carried out in the presence of control (QT0), 5 (QT5), 10 (QT10), and 20 (QT20) μg/mL of QT. Nuclear maturation, intracellular GSH and ROS content were evaluated following the IVM. In these oocytes, we subsequently evaluated the effect of QT supplementation on embryo development, including 2-cell, 8-cell, and blastocyst rate. The results of the present study showed that the supplementation of 10 μg/mL QT in maturation medium increased the number of MII oocytes. In addition, fertilization and blastocyst rate in QT10 treatment group were significantly higher in comparison to the other groups, and elevated the amount of intracellular GSH content compared to other QT concentrations and control groups. The intracellular ROS level was the lowest among oocytes matured in Q5 and Q10 treatment groups. This result suggested that quercetin dose-dependently improves nuclear maturation and embryo development, via reducing intracytoplasmic oxidative stress in mature oocyte.

Download Full-text

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab313 ◽

2015 ◽

Vol 27 (1) ◽

pp. 245 ◽

Cited By ~ 1

Author(s):

N. W. Santiquet ◽

A. F. Greene ◽

W. B. Schoolcraft ◽

R. L. Krisher

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Subsequent Development ◽

Developmental Competence ◽

Nuclear Maturation ◽

Oocyte Developmental Competence ◽

Oocyte Collection ◽

Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

Download Full-text

Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling

Cells ◽

10.3390/cells9061550 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1550 ◽

Cited By ~ 1

Author(s):

Marwa El Sheikh ◽

Ahmed Atef Mesalam ◽

Muhammad Idrees ◽

Tabinda Sidrat ◽

Ayman Mesalam ◽

...

Keyword(s):

Oxidative Stress ◽

In Vitro Maturation ◽

Inner Cell Mass ◽

Cell Mass ◽

Akt Signaling ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Vitamin B3 ◽

Cell Stage

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8–16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.

Download Full-text