scholarly journals Prematurational Culture with 3-Isobutyl-1-methylxanthine Synchronizes Meiotic Progression of the Germinal Vesicle Stage and Improves Nuclear Maturation and Embryonic Development in In Vitro-grown Bovine Oocytes

2014 ◽  
Vol 60 (1) ◽  
pp. 9-13 ◽  
Author(s):  
Weiping HUANG ◽  
Masashi NAGANO ◽  
Sung-Sik KANG ◽  
Yojiro YANAGAWA ◽  
Yoshiyuki TAKAHASHI
Keyword(s):  
Embryonic Development ◽  
Germinal Vesicle ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Bovine Oocytes

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

2018 ◽  
Vol 30 (1) ◽  
pp. 224
Author(s):  
L. M. S. Simoes ◽  
A. P. C. Santos ◽  
E. A. Lima ◽  
R. E. Orlandi ◽  
M. P. Bottino ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Causative Factor ◽  
Nuclear Maturation ◽  
Preovulatory Follicles ◽  
Fertilization Capacity ◽  
Bovine Oocytes ◽  
Metaphase I

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P < 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

180 LONG-TERM TRANSPORTATION OF BOVINE OOCYTES WITH MEIOTIC BLOCKERS: EFFECTS ON NUCLEAR MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 198
Author(s):  
P. C. Dall'Acqua ◽  
B. C. S. Leao ◽  
N. A. S. Rocha-Frigoni ◽  
G. B. Nunes ◽  
M. Ambrogi ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Study Data ◽  
Receptor Protein ◽  
Nuclear Maturation ◽  
Meiotic Inhibitors ◽  
Bovine Oocytes

The aim of this study was to assess the blockade and the reversal of meiosis block in bovine oocytes treated with a cyclin-dependent kinase inhibitor (butyrolactone-I; BL) combined or not with a selective inhibitor of epidermal growth factor receptor protein (tyrphostin AG 1478; AG) in a prematuration (PM) culture during oocyte transport. Cumulus-oocyte complexes (n = 4107) were transported in PM medium (TCM-199 with bicarbonate and 0.3% BSA) supplemented with one of the following inhibitors: 50 µM BL; 100 µM BL; 1 µM AG; 50 µM BL + 1 µM AG; or 100 µM BL + 1µM AG. Cumulus-oocyte complexes were transported in well-sealed polystyrene tubes (30 oocytes/tube) containing 200 μL of PM medium covered with mineral oil and gassed with 5% O2, 5% CO2, and 90% N2. The tubes were packed in a portable incubator (Thawing Unit MT 35/42, Minitub, Tiefenbach, Germany) at 38.5°C for 22 h. Afterward, treated oocytes were removed from meiotic inhibitors, transferred to in vitro maturation (IVM) medium (TCM-199 with bicarbonate, 0.5 mg mL−1 of FSH, 100 IU mL−1 of hCG, and 10% FCS), and cultured in a bench-top incubator (Thermo Fisher Scientific, Waltham, MA, USA) under 38.5°C and 5% CO2 in air for 20, 22, 24, or 26 h. The control groups were IVM for 20, 22, 24, or 26 h in IVM medium in the bench-top incubator at 38.5°C and 5% CO2 in air (Control; C) or in the portable incubator under the same conditions used for the treated groups (Transport Control; TC). For meiosis evaluation, oocytes were stained with 1% Hoescht immediately after follicle removal (0 h), at 6 and 22 h of PM, and after 20, 22, 24, and 26 h of IVM, and were classified as immature (germinal vesicle; GV) or mature (metaphase II; MII); intermediate phases of meiosis (GV breakdown, metaphase I, anaphase I, or telophase I) were not demonstrated in this study. Data were analysed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as mean ± standard error of the mean. The GV rates after 6 h of transport did not differ (P > 0.05) between 0-h oocytes (88.6 ± 2.3%) and the treated groups (70.3 ± 1.9% to 79.3 ± 2.2%); although GV rates of C (49.5 ± 2.4%) and TC (49.5 ± 2.4%) groups differed (P < 0.05) from 0-h oocytes, they did not differ from treated oocytes with the exception of the 1 µM AG group (79.3 ± 2.2%), which differed from TC (P < 0.05). After 22 h of transport, the GV rates of treated oocytes (50.3 ± 5.5 to 70.3 ± 6.6%) did not differ (P > 0.05) from 0-h oocytes (88.6 ± 2.3%) and were higher (P < 0.05) than C (4.6 ± 2.8%) and TC (8.3 ± 4.5%) that had the highest MII rates (68.4 ± 5.3 and 75.5 ± 2.0%, respectively, for C and TC) compared with the other groups (0 to 13.2 ± 10.2%). After meiotic inhibitors removal and IVM, meiosis block was fully reversed and there were no differences (P > 0.05) in the rates of MII between treated oocytes and C and TC groups after 20 (56.6%, averaged), 22 (57.7%, averaged), 24 (66.2%, averaged), or 26 h of IVM (57.0%, averaged). In conclusion, the meiotic inhibitors were effective in maintaining the majority of treated oocytes in GV stage after 22 h of transport and the inhibitory effect was fully reverted after its removal. Research was supported by FAPESP and CAPES.

scholarly journals Anti-Aging Effect of Urolithin A on Bovine Oocytes In Vitro

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2048
Author(s):  
Élisa Fonseca ◽  
Carla Cruz Marques ◽  
Jorge Pimenta ◽  
Joana Jorge ◽  
Maria Conceição Baptista ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Harmful Effect ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Adult Females ◽  
Urolithin A ◽  
Oocyte Aging ◽  
Bovine Oocytes

Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells’ (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes (NFE2L2, NQO1, and mt-DN5) and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.

scholarly journals Effect of concentration and exposure period to butyrolactone I on meiosis progression in bovine oocytes

2006 ◽  
Vol 58 (3) ◽  
pp. 354-359 ◽  
Author(s):  
P.R. Adona ◽  
C.L.V. Leal
Keyword(s):  
Drug Concentration ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Exposure Period ◽  
Culture Period ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Kinetics Of

The effect of concentration and exposure period of bovine oocytes to butyrolactone I (BLI) on meiotic block and in vitro maturation (IVM) kinetics was studied. In experiment 1, all oocytes were at germinal vesicle stage (GV), after 6h in culture with 0, 50 and 100µM BLI. After 12h, all oocytes cultured with 50 and 100µM BLI remained in GV. After 24h, less oocytes were in GV with 50µM (82%) than with 100µM BLI (99%, P<0.05). In experiment 2, after 6h IVM, 93% of control oocytes (IVM only) were in GV, while treated oocytes (100µM BLI for 6, 12 or 24h prior to IVM) showed less oocytes in GV with increased exposure period to BLI prior to IVM (83 and 73%, for 6h and 12h, P<0.05). For a 24h inhibition, GV rates were similar to 12h (70%, P>0.05). After 18h IVM, metaphase II (MII) rates were similar for all groups (76-81%). In experiment 3, after 6h IVM, 74% of treated oocytes (50 or 100µM BLI for 12h) were in GV. This rate was lower than for control oocytes (97.3%, P<0.05). After 18h IVM more oocytes (~80%, P>0.05) were in MII with BLI than for control (73%, P<0.05). Shorter culture periods require lower BLI concentration for meiotic block; initial nuclear maturation kinetics of oocytes cultured with BLI is accelerated, and this is affected by culture period but not by drug concentration.

Asynchronous meiotic progression in porcine oocytes matured in vitro: a cause of polyspermic fertilization?

1997 ◽  
Vol 9 (2) ◽  
pp. 187 ◽  
Author(s):  
Christopher G. Grupen ◽  
Hiroshi Nagashima ◽  
Mark B. Nottle
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Meiotic Progression ◽  
Development In Vitro ◽  
Two Populations

The progression of meiosis in porcine oocytes maturedin vitro was examined and the effect of maturation interval on the incidence of polyspermic fertilization and embryonic development in vitro was investigated. In Experiment 1, it was found that oocytes selected for in vitro maturation varied considerably in terms of their meiotic progression. Approximately half of the oocytes were already undergoing germinal vesicle breakdown at the start of maturation while the remainder were at the intact germinal vesicle stage. These two populations of oocytes developed to the metaphase-II stage by 24 h and 36 h of maturation respectively. Maturation for 40 h and 44 h did not increase the percentage of oocytes at metaphase-II stage observed at 36 h. In Experiment 2, reducing the maturation interval from 44 h to 36 h did not affect the percentage of oocytes penetrated but did decrease the rate of polyspermic fertilization (5% v. 34%; P < 0 ·05). In Experiment 3, development of the embryos produced in vitro was assessed after culture for seven days. Reducing the maturation interval from 44 h to 36 h decreased the percentage of embryos developing to the 8-cell (9% v. 18%; P < 0 · 05), morula (3% v. 10%; P < 0 · 01), and blastocyst (1% v. 8%; P < 0· 001) stages. These results suggest that maturation for 44 h gives rise to a population of ‘aged’ oocytes which is susceptible to polyspermic fertilization.

200 EFFECTS OF MELATONIN ON REACTIVE OXYGEN SPECIES GENERATION AND ACQUISITION OF EMBRYONIC DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 231
Author(s):  
P. C. Dall'Acqua ◽  
B. C. S. Leão ◽  
N. A. S. Rocha-Frigoni ◽  
M. Ambrogi ◽  
G. Z. Mingoti
Keyword(s):  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Signal Intensity ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Control Group ◽  
Oxygen Species ◽  
Nuclear Maturation ◽  
Intracellular Ros

Reactive oxygen species (ROS) are produced under normal culture conditions, but when production increases, it generates a harmful condition called oxidative stress (OS), leading to apoptosis and developmental blocks. Addition of antioxidants as melatonin to culture media has been used to minimize the effects of OS. Our hypothesis was that melatonin could improve oocyte in vitro maturation (IVM) and protect oocytes from ROS under a standard culture condition, thus increasing embryonic development. To test, cumulus-oocyte complexes were matured in TCM-199 with bicarbonate, 0.5 mg mL–1 of FSH, 100 IU mL–1 of hCG, and 10% FCS without supplementation (control group) or supplemented with 10–5 (MT5), 10–7 (MT7) or 10–9 (MT9) M melatonin for 22 h at 38.5°C and 5% CO2 in air. After IVM, a sample of oocytes (control, n = 59; MT5, n = 64; MT7, n = 77; MT9, n = 57) was stained with 1 µg mL–1 Hoechst 33342 to assess the nuclear maturation, and oocytes were classified as being in the stages of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I or telophase I (A/T), or metaphase II (MII). To determine the intracellular ROS levels, other matured oocytes (control, n = 46; MT5, n = 59; MT7, n = 51; MT9, n = 61) were stained with 5 µM CellROX®Green (Molecular Probes, Eugene, OR, USA) and were evaluated immediately under an epifluorescence inverted microscope (excitation 485 nm; emission 520 nm). The images were recorded and further analysed by Q-Capture Pro image software. After subtraction of the background signal intensity from the measured fluorescent signal intensity values, 1 group was chosen as a calibrator (control group), and each treatment value was divided by the mean calibrator value to generate the relative expression level (in arbitrary fluorescence units). Finally, another sample of matured oocytes (control, n = 188; MT5, n = 173; MT7, n = 180; MT9, n = 178) was submitted to IVF (Day = 0), and the presumptive zygotes were cultured in SOF at 38.5°C and 5% CO2 in air, for up to 7 days. The cleavage rates and embryonic development were evaluated at Days 3 and 7 of IVC, respectively. Data were analysed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as mean ± SEM. There was no effect (P > 0.05) of different concentrations of MT on nuclear status of matured oocytes, as we found no differences in the rates of GV (0% to 5.3% ± 3.4), GVBD (5.4% ± 3.2 to 16.3% ± 5.0), MI (1.7% ± 1.7 to 3.2% ± 3.2), AI/TI (0% to 5.4% ± 3.4), and MII (74.8% ± 2.7 to 87.5% ± 3.7). The cleavage rates did not differ (P > 0.05) among treatments (76.7% ± 4.4 to 83.8% ± 2.7), as well as the embryonic development to the blastocyst stage (31.2% ± 1.9 to 43.7% ± 5.7). The intracellular ROS levels decreased significantly (P < 0.05) in the MT9 group (0.75 ± 0.03) in comparison to Control (1.0 ± 0.06), MT5 (0.97 ± 0.05) and MT7 (0.94 ± 0.05). In conclusion, supplementation with 10–9 M melatonin during IVM reduced the intracellular ROS levels of oocytes without interfering with the nuclear maturation and the subsequent embryonic development to the blastocyst stage. Financial support was provided by FAPESP (#2013/07382–6).

182 EFFECTS OF IGF-1 OR IGF-1-LongR3 ON CELLULAR AND MOLECULAR ASPECTS OF CUMULUS-OOCYTE COMPLEXES DURING IN VITRO OOCYTE MATURATION IN CATTLE

2016 ◽  
Vol 28 (2) ◽  
pp. 222
Author(s):  
M. S. Araujo ◽  
M. D. Guastali ◽  
A. C. S. Castilho ◽  
F. Landim-Alvarenga
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Synthetic Analogue ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Metaphase Stage ◽  
Bovine Oocytes

The insulin-like growth factor-1 recombinant -3 (IGF-1-LongR3), a synthetic analogue of IGF-1 with increased bioavailability has not yet been used in vitro maturation (IVM) medium of bovine oocytes. Therefore, the aim of this study was to evaluate and compare the addition effects of IGF-1-LongR3 or IGF-1 in IVM bovine oocytes on meiotic progression, apoptosis, and profile of oocytes genes (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGFBP4 and IGFBP5) and genes in cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). Bovine ovaries were collected in slaughterhouses, and 739 oocytes with grades 1 or 2 were selected after aspiration of 2- to 8-mm follicles. IVM was carried out in TCM199 with FSH, LH, and antibiotics (BM) supplemented with 100 ng mL–1 IGF-1 or 100 ng mL–1 LongR3-IGF-1. Control oocytes were matured in BM supplemented with 0.1% polyvinyl alcohol (PVA) or 10% FCS. For all groups, maturation was performed during 22–24 h in an incubator at 38.5°C and 5% CO2 in air. Subsequently oocytes were denuded and analysed for apoptosis, nuclear maturation, and gene expression by TUNEL assay, staining Hoechst 33342, and RT-qPCR, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyse the results obtained by RT-qPCR. After 10 replicates of IVM, 339 oocytes were evaluated for meiotic progression and apoptosis and 400 oocytes for gene expression. There was no statistical difference between the experimental groups with respect to meiotic progression and apoptosis. BCL2 and IGFBP4 gene were less expressed in oocytes matured with IGF-1 and LongR3-IGF-1 compared with control groups. GFBP4 was also less expressed in cumulus cell of oocytes from the experimental groups. Moreover COX2 expression was statistically elevated in cumulus cells matured in the presence of IGF-1 and LongR3-IGF-1 It was possible to perform IVM of bovine oocytes in the presence of LongR3-IGF-1, allowing its use in replacement of IGF-1 and FCS. The results of this study will provide more information on the interaction of IGF with the IGFBP and its importance for oocyte maturation.

scholarly journals Meiotic inhibition of bovine oocytes in medium supplemented with a serum replacer and hormones: effects on meiosis progression and developmental capacity

Zygote ◽  
2010 ◽  
Vol 19 (2) ◽  
pp. 107-116 ◽  
Author(s):  
Letícia Siqueira Sá Barretto ◽  
Viviane Sgobbi Dias Caiado Castro ◽  
Joaquim Mansano Garcia ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Nuclear Maturation ◽  
Developmental Capacity ◽  
Bovine Oocytes ◽  
Kinetics Of ◽  
Hatching Rates

SummaryAiming to improve the developmental competence of bovine oocytes during meiotic block, this study evaluated the effects of a serum replacer (Knockout SR®) and hormones (gonadotropins and estradiol) supplementation of prematuration medium (TCM119 with 0.5 mM IBMX [IBMX group] or 25 μM roscovitine [ROSC group]) on the kinetics of oocyte nuclear maturation and embryo development. Most IBMX and ROSC oocytes prematured for 8 h culture remained in the GV stage (70.3% and 73.1%, respectively; p > 0.05) similar to Control 8 h (63.5%) and to control immature oocytes (Control 0 h, 92.5%). After prematuration for 16 h, no oocytes remained in the GV stage at similar rates to those recently aspirated (p < 0.05); GV rates in ROSC (32.4%) were higher (p < 0.05) than in the Control 16 h group (8.6%), but similar (p > 0.05) to IBMX (9.7%). After in vitro maturation (IMV) for 24 h, metaphase II (MII) rates for oocytes prematured during 8 h were similar (p > 0.05) between control and treatments (65.0–71.7%). Similarly, MII rates oocytes prematured during 16 h were similar (p > 0.05) between all groups (45.9–60.4%). Cleavage rates (67.8–78.2%), embryonic development in day-7 (25.0–35.6%) and hatching rates in day-8 (2.5–11.3%) oocytes blocked during 8 h were similar for all groups (p > 0.05). Results indicate that addition of Knockout SR® and hormones to meiotic block culture with IBMX and roscovitine negatively affected meiotic arrest, but did not impair oocyte nuclear maturation and acquisition of developmental competence.

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