199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
G. Z. Mingoti ◽  
F. Filion ◽  
P. Vincent ◽  
L. C. Smith
Keyword(s):  
Cell Communication ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Additional Time ◽  
Chi Square ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

scholarly journals 319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

2004 ◽  
Vol 16 (2) ◽  
pp. 279
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
F. Prati ◽  
G. Mari
Keyword(s):  
Polar Body ◽  
Basal Medium ◽  
Defined Medium ◽  
Chi Square ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Chi Square Test ◽  
The Media ◽  
Cytoplasmic Maturation

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P<0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P<0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽  
2018 ◽  
Vol 26 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Mohamed Fathi ◽  
A. Salama ◽  
Magdy R. Badr
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Free Medium ◽  
Maturation Time ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

scholarly journals cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2512
Author(s):  
Daniela-Alejandra Medina-Chávez ◽  
Irene Sánchez-Ajofrín ◽  
Patricia Peris-Frau ◽  
Carolina Maside ◽  
Vidal Montoro ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Maturation ◽  
Phosphodiesterase Inhibitor ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Assisted Reproduction Technologies ◽  
Underlying Mechanisms

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

49 Developmental Potential of Dromedary Camel Oocytes Vitrified at the Germinal Vesicle Stage: Effects of Different Cryoprotectant Combinations and Cryo-Carriers

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
M. Fathi ◽  
A. R. Moawad ◽  
M. R. Badr
Keyword(s):  
Survival Rates ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
And Control ◽  
Vitro Production

Cryopreservation of oocyte would be an alternative to overcome the limited availability of dromedary camel oocytes and allow improvements in in vitro production in this species. Our aim was to develop a protocol for vitrification of dromedary camel oocytes at the germinal vesicle (GV) stage using various cryoprotectant combinations and cryo-carriers. In experiment 1, cumulus–ppcyte complexes (COC) obtained at slaughter were equilibrated in a solution composed of 10% ethylene glycol (EG) and 0.25 M trehalose. The oocytes were then exposed for 60 s to vitrification solutions (VS) composed of 20% EG and 20% dimethyl sulfoxide (DMSO; VS1) or 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3). The COC were then transferred into decreasing concentration of trehalose solution (toxicity test). In experiment 2, COC were randomly divided into 4 groups and vitrified by using straw or open pulled-straw (OPS) or solid surface vitrification (SSV) or cryotop in VS1 or VS2. Following vitrification and warming viable oocytes were matured in vitro for 30 h at 39°C in 5% CO2 in air. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels and then cultured in modified KSOMaa medium for 7 days. Oocyte viability, maturation, fertilization, and embryo development were evaluated. Data were analysed using one-way ANOVA and t-test. Viability and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%) than those exposed to VS1 (68.2% and 48.0%) and VS2 (79.3% and 56.9%). Although recovery rates were significantly lower (P ≤ 0.05) in oocytes vitrified using SSV or cryotop in either VS1 or VS2 solutions (66.9% to 71.1%) than those vitrified by straws using VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in SSV and cryotop groups (90.7% to 94.8%) than straw and OPS (68.2% to 86.5%) groups. Among vitrified groups, maturation and fertilization rates (51.8% and 39.2%, respectively) were the highest in the cryotop-VS2 group. Those values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and cryotop groups than in straws. No significant differences were observed in these parameters between cryotop and control groups. Together, the results show that both vitrification solution and cryodevice affect viability and developmental competence of vitrified/warmed dromedary camel oocytes. We report for the first time that dromedary camel oocytes vitrified at the GV stage have the ability to be matured, fertilized, and subsequently develop in vitro to produce blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

scholarly journals Glial Cell Line-Derived Neurotrophic Factor: An Intraovarian Factor that Enhances Oocyte Developmental Competence in Vitro

Endocrinology ◽  
2007 ◽  
Vol 148 (9) ◽  
pp. 4292-4301 ◽  
Author(s):  
Katja Linher ◽  
De Wu ◽  
Julang Li
Keyword(s):  
Cell Line ◽  
Glial Cell ◽  
Neurotrophic Factor ◽  
Preimplantation Embryo ◽  
Cyclin B1 ◽  
Developmental Competence ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

The success of early embryonic development depends on oocyte nuclear and cytoplasmic maturation. We have investigated whether glial cell line-derived neurotrophic factor (GDNF) affects the in vitro maturation (IVM) of porcine oocytes and their subsequent ability to sustain preimplantation embryo development. GDNF and both its coreceptors, GDNF family receptor α-1 (GFRα-1) and the rearranged during transformation (RET) receptor, were expressed in oocytes and their surrounding cumulus cells derived from small and large follicles. When included in IVM medium, GDNF significantly enhanced cumulus cell expansion of both small and large cumulus-oocyte complexes and increased the percentage of small follicle-derived oocytes maturing to the metaphase II stage, although nuclear maturation of large oocytes was not significantly affected. Examination of cyclin B1 protein expression as a measure of cytoplasmic maturation revealed that in the presence of GDNF, cyclin B1 levels were significantly increased in large follicle-derived oocytes, as well as in oocytes from small follicles to a level comparable to the untreated large group. After activation, a significantly higher percentage of both small and large oocytes that were matured in the presence of GDNF developed to the blastocyst stage compared with untreated controls. Indeed, GDNF enhanced the blastocyst rate of small oocytes to levels comparable to those obtained for large oocytes matured without GDNF. The effect of GDNF was specific; this was evident because its enhancement of nuclear maturation and embryo developmental potential was blocked by an antibody against GFRα-1. Our study provides the first functional evidence that GDNF affects oocyte maturation and preimplantation embryo developmental competence in a follicular stage-dependent manner. This finding may provide insights for improving the formulation of IVM culture systems, especially for oocytes from small follicles.

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 The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium

2021 ◽  
Vol 22 (20) ◽  
pp. 11148
Author(s):  
Radek Procházka ◽  
Alexandra Bartková ◽  
Lucie Němcová ◽  
Matej Murín ◽  
Ahmed Gad ◽  
...  
Keyword(s):  
Time Course ◽  
Cumulus Cell ◽  
Cell Communication ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Control Medium ◽  
Developmental Potential ◽  
Akt Activation ◽  
Expression Of Genes

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

356 MODULATION OF PORCINE NUCLEAR MATURATION STATUS USING A SPECIFIC PHOSPHODIESTERASE INHIBITOR

2010 ◽  
Vol 22 (1) ◽  
pp. 334
Author(s):  
M. L. Sutton-McDowall ◽  
R. B. Gilchrist ◽  
J. G. Thompson
Keyword(s):  
Gap Junction ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Adenosine Monophosphate ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Gap Junction Communication ◽  
Defined Culture ◽  
Type 3

Compared to other livestock species, IVM porcine COCs have poor developmental competence.Thisismost likely due to poor cytoplasmic maturation and asynchronous nuclear maturation. The resumption of nuclear maturation is largely regulated by cyclic adenosine monophosphate(cAMP), with increasing intra-oocyte levels prolonging cumulus-oocyte gap junction communication and delaying meiotic resumption. Modulation of cAMP levels using phosphodiesterase (PDE) inhibitors during bovine IVM significantly improves developmental competence (Thomas et al. 2004 Biol. Reprod. 71, 1142-1149). Hence, the aim of this study was to determine the effect of a type 3 PDE inhibitor (cilostamide, CIL) supplementation during porcine IVM on nuclear maturation, using a defined culture system. COCs derived from follicles of prepubertal gilts were cultured in groups of 10 in 100μL IVM medium (VitroMat, IVF Vet Solutions, Adelaide, Australia) +100 m IU mL-1 rhFSH +4 mg mL-1 BSA and nuclear maturation was assessed using orcein dye. Exp. 1: IVM medium was supplemented with 0, 5, 10, and 20 μM CIL or 0, 0.01, 0.1, 1, and 10 μM CIL and nuclear maturation was determined at 24 h and 44 h. Exp. 2: COCs were cultured in ± 0.1 μM CIL and nuclear maturation was assessed at 24 h, 44 h, 48 h, 52 h, and 56 h. Proportions of COCs at each stage of nuclear maturation were arcsine transformed and differences determined using a general linear model and Bonferroni post hoc test. Four replicates per experiment were performed with 20 COCs used for each treatment and/or time point. Results for Exp. 1 revealed no differences in the rate of nuclear maturation after 24 h of culture. After 44 h, 77% of COCs incubated in the absence of CIL were at metaphase II (MII) compared to 35-45% MII when cultured in the presence of 1, 5, 10, or 20 μM CIL (P < 0.001). Furthermore, CIL supplementation resulted in approximately half the COCs arresting at germinal vesicle stage (GV) after 44 h of culture. While there were no significant differences in the MII rates of COCs cultured in 0, 0.01, and 0.1 μM CIL, significantly more COCs were at metaphase I (MI) at 44 h compared to control COCs (0 μM CIL, P < 0.05). The time course experiment (Exp 2) demonstrated that nuclear maturation was delayed by 12 h with 0.1 μM CIL, compared to the absence of CIL, with comparable MII rates being achieved at 56 h for 0.1 μM CIL (72%) and 44 h for controls (0 μM CIL = 73%). These results demonstrate that porcine oocyte maturation can be induced in vitro by FSH in the presence of a low dose of type 3 PDE inhibitor resulting in a delay in the resumption and completion of nuclear maturation. Further investigations are underway to determine if CIL treatment prolongs gap junction communication and improves the developmental competence of porcine oocytes. This work was supported by the National Institutes of Health (USA) and National Health and Medical Research Council (Australia).

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