scholarly journals Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by prostaglandins E2 and F2α

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 733-740 ◽  
Author(s):  
Marcos H Barreta ◽  
João Francisco C Oliveira ◽  
Rogério Ferreira ◽  
Alfredo Q Antoniazzi ◽  
Bernardo G Gasperin ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Gnrh Agonist ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Similar Rate ◽  
Follicular Cells ◽  
Lh Surge ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Prostaglandins E2 And F2α

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 μM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%;P<0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 μM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin;P<0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE2, PGF2αor AngII was present in the co-culture system with follicular cells (PGE277.4%, PGF2α70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE2or PGF2αproduced by follicular cells.

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.

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.

Differential effects of linoleic and alpha-linolenic fatty acids on spatial and temporal mitochondrial distribution and activity in bovine oocytes

2012 ◽  
Vol 24 (5) ◽  
pp. 679 ◽  
Author(s):  
Waleed F. Marei ◽  
D. Claire Wathes ◽  
Ali A. Fouladi-Nashta
Keyword(s):  
Membrane Potential ◽  
Oocyte Maturation ◽  
Inhibitory Effect ◽  
Inner Membrane ◽  
Nuclear Maturation ◽  
Mitochondrial Inner Membrane ◽  
Maturation Rate ◽  
Microscope Imaging ◽  
Mitochondrial Distribution ◽  
Bovine Oocytes

Using specific stains and confocal microscope imaging, the patterns of mitochondrial distribution, mitochondrial inner membrane potential and reactive oxygen species (ROS) levels during bovine oocyte maturation were determined in the presence or absence of physiological concentrations of linoleic acid (LA; 100 µM) or α-linolenic acid (ALA; 50 µM). Mitochondrial distribution in control oocytes at 0 h was mainly peripheral and changed to a diffused pattern after 1 h of culture; this was maintained up to 24 h. Mitochondrial clusters were observed during the early hours of maturation (1–4 h); the majority of these were arranged in perinuclear fashion. LA supplementation resulted in: (1) delayed redistribution of the mitochondria from a peripheral to a diffuse pattern and a decreased percentages of oocytes showing perinuclear mitochondrial clusters, (2) decreased mitochondrial inner membrane potential at 1 and 24 h compared with the control and (3) higher ROS levels, associated with a lower nuclear maturation rate. In contrast, ALA supplementation had no effect on mitochondrial distribution and activity and decreased ROS levels compared with the control; this was associated with an increased nuclear maturation rate. In conclusion, LA induced alterations in mitochondrial distribution and activity as well as increasing ROS levels, which mediate, at least in part, the inhibitory effect on oocyte maturation.

167 Effect of Storage Time and Temperature of a Commercial Embryo Holding Medium on the Maturation Kinetics of Immature Bovine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 223
Author(s):  
O. B. Pascottini ◽  
M. Catteeuw ◽  
A. Van Soom ◽  
G. Opsomer
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Holding Time ◽  
Control Group ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Maturation Stages ◽  
Bovine Oocytes ◽  
Kinetics Of

The effect of holding time and temperature during storage of immature bovine oocytes in a commercial embryo holding medium (EHM; Syngro® Ltd., Livingston, United Kingdom) was evaluated. Ovaries were collected at the local slaughterhouse and processed within 2 h. Cumulus-oocyte complexes (COC) were collected and allocated to groups of 60. The COC were held in 1-mL sterile glass osmometer tubes, filled to the top with the EHM to limit the amount of air. Vials were capped and covered with parafilm to ensure a tight seal and prevent leakage. Tubes were stored for 6 h at 4°C, room temperature (RT), or 38.5°C; for 10 h at 4°C and RT; and for 14 h at RT. Next, oocytes were fixed after storage in EHM (immature holding) or fixed after being held in EHM and subsequent 22-h maturation at 38.5°C in 5% CO2 in humidified air (mature holding). Maturation medium consisted of modified bicarbonate-buffered TCM-199 supplemented with gentamycin and epidermal growth factor. During all experiments, a control group was included each time. The control consisted of groups of 60 COC immediately fixed after collection or transferred to maturation medium for 22 h and subsequently fixed. Nuclear maturation of oocytes was assessed after Hoechst 33342 staining, using a 400× magnification fluorescence microscope. A total of 3043 COC were evaluated in 3 replicates. Oocytes maturation stages were classified as (1) oocytes in germinal vesicle stage, (2) oocytes in meiotic progression (diakinesis, metaphase I, or anaphase), (3) matured (telophase I or metaphase II), and (4) degenerated (degraded chromatin). Oocytes remained at the germinal vesicle stage when held in EHM (without subsequent maturation) regardless of holding time and temperature (P > 0.05). When oocytes were held for 6 h and subsequently matured (Table 1), the number of matured oocytes was significantly lower for oocytes held at 38.5°C compared with the other groups (control, RT, and 4°C). When held for 10 h, the oocyte maturation rate was similar between the control and RT groups (P > 0.05), but it was significantly lower in oocytes held at 4°C. Last, when compared with oocytes held at RT for 14 h, the maturation rate was higher in the control group (P < 0.05). To conclude, immature bovine oocytes can be successfully held in EHM at RT for up to 10 h. Storing immature oocytes in EHM can delay oocyte maturation and concomitantly synchronize maturation. Table 1.Kinetics of cumulus-oocyte complex nuclear status after storage in embryo holding medium for different times and temperatures and subsequent 22-h maturation

Manganese inhibits spontaneous nuclear maturation in bovine cumulus-enclosed oocytes

2001 ◽  
Vol 81 (2) ◽  
pp. 223-228 ◽  
Author(s):  
Sylvie Bilodeau-Goeseels
Keyword(s):  
Follicular Fluid ◽  
Germinal Vesicle ◽  
Atomic Absorption Spectrophotometry ◽  
Inhibitory Effect ◽  
Manganese Concentration ◽  
Maturation Stage ◽  
Bovine Oocyte ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Oocyte Meiosis

The objective of this work was to examine the effects of manganese concentration on nuclear maturation of bovine cumulus-enclosed (CEO) and denuded oocytes cultured for 7 h or 22 h. Following culture, oocytes were then fixed and stained for assessment of nuclear maturation stage. The addition of MnCl2 significantly suppressed nuclear maturation after 7h of culture (15, 69, 84 and 70% of oocytes were still at the germinal vesicle stage after culture with 0, 50 μM, 0.5 mM and 5mM MnCl2, respectively; P < 0.001). However, MnCl2 was without significant effect on denuded oocytes cultured for 7 h. When CEO and denuded oocytes were cultured with manganese for 22 h, the percentages of mature oocytes were reduced (96 , 20, 15, 3% and 80, 39, 53 and 16% for CEO and denuded oocytes cultured with 0, 50 μM, 0.5 mM and 5 mM MnCl2, respectively; P < 0.0005). The inhibitory effect of 50 μM MnCl2 was transient and reversible because it did not maintain oocytes in meiotic arrest after 22 h of culture. In addition, 72% of the CEO cultured with 50 μM MnCl2 for 7 h and subsequently cultured without manganese for 18 h were mature. The concentration of manganese (6 ± 1 μM) in follicular fluid (as determined by atomic absorption spectrophotometry) was below inhibitory concentrations. In conclusion, manganese inhibited germinal vesicle breakdown in bovine CEO; however, only the effect of the lowest concentration tested (50 μM) was reversible. Key words: Bovine, oocyte, meiosis, manganese, follicular fluid

Follicular cells affect the fertilizability and developmental competency of bovine oocytes in vitro

1997 ◽  
Vol 9 (8) ◽  
pp. 763 ◽  
Author(s):  
K. S. Kim ◽  
N. Minami ◽  
M. Yamada ◽  
K. Utsumi
Keyword(s):  
Subsequent Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Follicular Cells ◽  
Maturation Period ◽  
Nuclear Maturation ◽  
Maturation In Vitro ◽  
Further Development ◽  
Bovine Oocytes

The present study examined the time-dependent effects of follicular cells on the fertilizability of oocytes and their subsequent development to blastocysts. The percentages of oocytes reaching the metaphase-II stage of maturation rose from 51·3% after 16 h of culture to 86·2% at 28 h (cumulus-intact oocytes; CIO) and, for the same time points, from 65·4% to 83·3% (corona-enclosed oocytes; CO) and 54·3% to 88·9% (denuded oocytes; DO), respectively. When DO were cultured for more than 24 h before insemination, fertilization rates were significantly lower compared with CIO and CO. The maximum rates of development to blastocysts were observed when the oocytes were cultured for 24 h in the CIO group (22·1%), 20 h in the CO group (19· 7%) and 18 h in the DO group (9·2%), respectively. These results suggest that (i) the presence of cumulus cells or corona cells during maturation is not necessary for nuclear maturation of oocytes; (ii) the attachment of corona cells to the oocytes during maturation is important for the further development to the blastocyst stage, and (iii) the presence of attached cumulus and/or corona cells during maturation in vitro extends the maturation period required for further development to the blastocyst stage.

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.

scholarly journals 274 NUCLEAR MATURATION KINETICS AND IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES TREATED WITH BUTYROLACTONE I COMBINED OR NOT WITH ROSCOVITINE

2005 ◽  
Vol 17 (2) ◽  
pp. 287
Author(s):  
P.R. Adona ◽  
M.D. Quetglas ◽  
P.R.L. Pires ◽  
C.L.V. Leal
Keyword(s):  
Embryo Development ◽  
Kinase Inhibitors ◽  
Point Group ◽  
Germinal Vesicle ◽  
Oocyte Competence ◽  
Cell Numbers ◽  
Nuclear Maturation ◽  
Developmental Rates ◽  
Bovine Oocytes

Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696–1701). The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579–585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 μM BLI (B) or 6.25 μM BLI + 12.5 μM ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 μg mL−1 FSH, 50 μg mL−1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40–50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL−1 EGF). After IVM, all groups (140–150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 × 106 sperm cells mL−1) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5°C under 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 ± 1%, P < 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 ± 2 and 80 ± 1%, respectively; P > 0.05) than C (100%, P < 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 ± 1–83 ± 2%, P > 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 ± 1–89 ± 1 for 21 h and 85 ± 2–96 ± 8 for 24 h P > 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 ± 3–84 ± 3%, P > 0.05) and Day 7 blastocyst rates (26 ± 4–37 ± 4%, P > 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 ± 4–40 ± 4%, P > 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 ± 4%) only when compared with C (40 ± 4%, P < 0.05). Hatching rates were similar (10 ± 3–16 ± %3, P > 0.05) as were total cell numbers (141 ± 5–170 ± 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in about 3 h; and (c) BR used in DMEM during prematuration may negatively affect developmental rates. Financial support was provided by Fapesp, Brazil.

scholarly journals Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro

Reproduction ◽  
2005 ◽  
Vol 129 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Pilar Coy ◽  
Raquel Romar ◽  
Rebecca R Payton ◽  
Lisa McCann ◽  
Arnold M Saxton ◽  
...  
Keyword(s):  
Embryo Development ◽  
Lens Culinaris ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cortical Granule ◽  
Nuclear Maturation ◽  
Development In Vitro ◽  
Hoechst Staining ◽  
Bovine Oocytes

The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus–oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 μmol/l S-roscovitine for 24 h. Hoechst staining showed that 50 μmol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 μmol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin–fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 μmol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8–16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

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