79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 178
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Nile Red ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Treatment Groups ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Conventional Freezing

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

2017 ◽  
Vol 29 (1) ◽  
pp. 129 ◽  
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Equilibration Time ◽  
Step Size ◽  
A Cell ◽  
Blastocyst Rate ◽  
Mitotracker Red ◽  
Main Effects ◽  
Conventional Freezing

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

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.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

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 Beneficial Effects of Neurotrophin-4 Supplementation During in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Embryonic Development After Parthenogenetic Activation

2021 ◽  
Vol 8 ◽  
Author(s):  
Mirae Kim ◽  
Seon-Ung Hwang ◽  
Junchul David Yoon ◽  
Joohyeong Lee ◽  
Eunhye Kim ◽  
...  
Keyword(s):  
Treatment Group ◽  
Signaling Pathway ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Developmental Competence ◽  
Treatment Groups ◽  
Beneficial Effects ◽  
Significant Difference ◽  
Egf Signaling

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75NTR) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.

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

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.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Oocyte Growth ◽  
North West ◽  
Significant Difference ◽  
Cytoplasmic Maturation

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

175 EFFECT OF ACETYL-CoA CARBOXYLASE (ACC) INHIBITOR ON THE LIPID CONTENT AND NUCLEAR MATURATION OF CANINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 202 ◽  
Author(s):  
J. McGill ◽  
G. Reddy ◽  
L. Simon ◽  
G. Wirtu
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Animal Health ◽  
Fluorescent Microscopy ◽  
Cumulus Cell ◽  
Nile Red ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nuclear Maturation

Compared with other domestic species, embryo technologies are least developed for the dog. This is mainly due to difficulties in producing mature oocytes in vitro. Canine oocytes contain exceptionally high amounts of lipid. High lipid content increases the chilling sensitivity of oocytes and embryos. Mechanical and chemical reductions of the lipid content have been used to improve the cryotolerance of oocytes. Additionally, chemical stimulation of lipid catabolism improved oocyte in vitro maturation (IVM) rates in other species (You et al. 2012 Theriogenology 78, 235–543). Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo lipogenesis and its expression has been reported in oocytes and embryos. In somatic cells, inhibition of ACC reduces lipogenesis and enhances β-oxidation. Our hypothesis is that treatment of oocytes with an inhibitor of ACC (CP640186, Pfizer Animal Health, New York, NY, USA) reduces lipid content and improves IVM rate of oocytes. Ovaries were collected from a spay clinic and sliced in HEPES-buffered TCM-199 to recover oocytes. In vitro maturation was conducted at 38.5°C, 5% CO2, and high humidity in TCM-199 supplemented with 1% fetal bovine serum, glutamine, sodium pyruvate, β-mercaptoethanol, oestradiol, epidermal growth factor, and antimicrobial agents (Songsasen et al. Mol. Reprod. Dev. 79, 186–196). During the first 19 to 21 h, the IVM media contained 4 concentrations of the inhibitor (0+DMSO, 0.02, 0.1, and 0.5 μM, designated as treatments 1, 2, 3, and 4, respectively) and then oocytes were transferred to a medium without the inhibitor and cultured for an additional 27 to 29 h. At the end of culture (total of 48 h), oocytes were denuded of cumulus layers, washed, fixed, and stained with Nile red (lipid) and Hoechst-33342 (chromatin), and then mounted on a microscope slide. Lipid content and chromatin status were evaluated using fluorescent microscopy (TRITC and DAPI filters, respectively). The relative lipid content was measured by the corrected total cell fluorescence (CTCF) using ImageJ software (http://rsbweb.nih.gov/ij/). Data on CTCF and proportions of chromatin status of oocytes were analysed using one-way ANOVA (SigmaPlot 11.0). The mean CTCF for each treatment was 5.5 × 109 (n = 51, 5.2 × 109 (n = 44), 4.5 × 109 (n = 31), and 4.8 × 109 (n = 34), respectively (P = 0.3; 4 replicates). At the highest dose, the agent induced relatively more cumulus cell layer expansion but inhibited their attachment to the dish; the latter effect was reversible because cumulus cells attached and proliferated after washing the oocytes of the agent. Metaphase II was rare (≤3.1%); however, the proportion of oocytes developing to ≥GVBD stage (Trt 1 14%, n = 37; Trt 2 41%, n = 56; Trt 3 5%, n = 22; Trt 4 11%, n = 43) was affected by treatments. Our preliminary data indicate that a low concentration of ACC inhibitor has a positive effect on the nuclear maturation of canine oocytes but the effect on lipid content as estimated by using Nile red fluorescence intensity appears to be minimal.

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