scholarly journals 293 TWO-STEP MATURATION OF BOVINE OOCYTES WITHOUT CDK INHIBITORS: AN ALTERNATIVE TO AFFECT THEIR SUBSEQUENT DEVELOPMENTAL COMPETENCE

2005 ◽  
Vol 17 (2) ◽  
pp. 297
Author(s):  
A. Pavlok ◽  
G. Lapathitis ◽  
S. Cech ◽  
M. Kubelka ◽  
M. Lopatarova ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Germinal Vesicle ◽  
Culture Conditions ◽  
Medium Composition ◽  
Developmental Competence ◽  
Second Step ◽  
Cyclin Dependent Kinase ◽  
Significant Difference ◽  
One Step ◽  
Bovine Oocytes

The acquisition of meiotic and developmental competence seems to correlate not only with the size of follicles and oocytes but also with the morphology and transcriptional activity of the oocyte nuclei and nucleoli. To secure or increase the fertilization and the developmental competence of bovine oocytes, we have developed a two-step culture system using the specific cyclin dependent kinase inhibitors (Butyrolactone I, Bohemine). However, these drugs have several side effects during the prolonged time of culture. To avoid this disadvantage, we have used in the present experiments modified culture conditions simulating the intrafollicular block of meiosis. In the first step of culture, bovine oocytes isolated from small, medium, and large follicles (2–3, 3–4, and 4–6 mm in diameter, respectively) were kept under conditions that secured for at least 48 h the intact germinal vesicle stage (GV) in more than 90% of oocytes. The second step represented the subsequent 20–22 h in conditions stimulating resumption of meiosis. The effectiveness of this model depended mainly on medium composition: reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity of a medium by agar (0.3%), and reduction of oxygen concentration (within 6–9%). The reduction of the proportion between the number of cumulus-oocyte complexes (COC) and the amount of medium (within 6–7 mL per COC) should amplify the GVBD-inhibiting effect of oocyte-surrounding granulosa cells. The COC were situated in clots of 6–7 COC per clot. The effectiveness and reversibility of GVBD inhibition depends also on the duration of COC isolation. The full reversibility of GVBD inhibition was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 h) maturation technique was evaluated by the percentage of total and hatched Day 9 blastocysts. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from the smallest follicular category (13.9 vs. 7.1% and 9.2 vs. 3.3% for total and hatched blastocysts, respectively). No significant difference was noticed between between one- and two-step culture when oocytes from large healthy follicles were used. However, the two-step maturation of oocytes from regressing follicles substantially reduced the blastocyst yield (9.7 vs. 39.1% and 4.9 vs. 26.7% for total and hatched blastocysts, respectively). This study was supported by grant of GA CR No. 524/02/0674.

Meiotic inhibition with different cyclin-dependent kinase inhibitors in bovine oocytes and its effects on maturation and embryo development

Zygote ◽  
2004 ◽  
Vol 12 (3) ◽  
pp. 197-204 ◽  
Author(s):  
Paulo Roberto Adona ◽  
Cláudia Lima Verde Leal
Keyword(s):  
Embryo Development ◽  
Kinase Inhibitors ◽  
Germinal Vesicle ◽  
Oocyte Activation ◽  
Cyclin Dependent Kinase ◽  
Nuclear Maturation ◽  
Inhibition Control ◽  
Inhibition Experiment ◽  
Bovine Oocytes

Cyclin-dependent kinase inhibitors (CDKIs) such as butyrolactone I (BL-I) and roscovitine (ROS) maintain bovine oocytes blocked at the germinal vesicle (GV) stage. Bohemine (BOH), another CDKI, has been used for oocyte activation. The objective of this study was to determine whether BOH blocks meiosis and to compare its efficiency with other CDKIs (ROS and BL-I). Oocytes were cultured for 24 h in 0, 50, 100 and 150 μM BOH to determine the best concentration for blocking meiosis (experiment 1). GV rates were 3.3%, 64.5%, 83.3% and 88.9% (0, 50, 100 and 150 μM, respectively). Experiment 2 compared meiotic inhibition efficiency of BOH (100 μM), ROS (25 μM) and BL-I (100 μM). BL-I presented the highest GV rates (97.5%). BOH and ROS were similar to each other (85.4% and 79.9%, respectively). To assess the reversibility of meiotic inhibition (experiment 3), oocytes underwent in vitro maturation (IVM) for 18 h after the 24 h inhibition. Control oocytes were submitted to IVM for 18 h (C18) or 24 h (C24). Maturation rates were either similar to (ROS and BL-I: 96.0% and 93.6%, respectively) or superior to (BOH, 96.9%) C24 (91.0%). All groups were superior to C18 (82.5%). In experiment 4, oocytes were treated as in experiment 3 and then in vitro fertilized and cultured for 8 days. Blastocyst rates for BL-I (32.3%) were similar to C24 (35.0%), while those for BOH (20.2%) and ROS (24.2%) were inferior. All groups were inferior to C18 (43.4%). The results show that: (a) BOH inhibits meiosis resumption; (b) BL-I is the most effective of the CDKIs tested for blocking meiosis; (c) culture of oocytes with meiosis inhibitors is fully reversible in terms of nuclear maturation but they may either decrease (BOH and ROS) or maintain (BL-I) embryo development rates.

scholarly journals Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg
Keyword(s):  
Dna Fragmentation ◽  
Oocyte Maturation ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
The Mean ◽  
Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

2018 ◽  
Vol 30 (1) ◽  
pp. 206
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
E. Shedova ◽  
A. Lopukhov ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Calf Serum ◽  
Developmental Competence ◽  
Second Step ◽  
Russian Science ◽  
Cleavage Rate ◽  
System 1 ◽  
And Control ◽  
Bovine Oocytes

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

scholarly journals A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle

2019 ◽  
Vol 31 (12) ◽  
pp. 1793 ◽  
Author(s):  
Valentina Lodde ◽  
Silvia Colleoni ◽  
Irene Tessaro ◽  
Davide Corbani ◽  
Giovanna Lazzari ◽  
...  
Keyword(s):  
Gap Junction ◽  
Embryo Development ◽  
Large Scale ◽  
Germinal Vesicle ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Morphological Criteria ◽  
Chromatin Configuration ◽  
Follicle Size ◽  
Meiotic Competence

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus–oocyte complexes (COCs) according to follicle size (&lt;1, 1–2, &gt;2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles &lt;1 and 1–2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1–2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P&lt;0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles &lt;1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

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.

Fertilization and Development of Frozen–Thawed Germinal Vesicle Bovine Oocytes by a One-Step Dilution Methodin Vitro

Cryobiology ◽  
1996 ◽  
Vol 33 (5) ◽  
pp. 515-524 ◽  
Author(s):  
T. Suzuki ◽  
A. Boediono ◽  
M. Takagi ◽  
S. Saha ◽  
C. Sumantri
Keyword(s):  
Germinal Vesicle ◽  
One Step ◽  
Bovine Oocytes

scholarly journals 288 RELATIONSHIP BETWEEN CHROMATIN ORGANIZATION AND OOCYTE - CUMULUS CELL COMMUNICATION IN GERMINAL VESICLE STAGE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 294
Author(s):  
V. Lodde ◽  
C. Galbusera ◽  
S. Modina ◽  
M.S. Beretta ◽  
A. Lauria ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hoechst 33342 ◽  
Oocyte Growth ◽  
The Status ◽  
Chromatin Configuration ◽  
Bovine Oocytes

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, and the progressive chromatin condensation has been related to the acquisition of embryonic developmental potential. However, little is known about the mechanisms that regulate chromatin remodeling. In immature mouse oocytes, chromatin condensation and redistribution around the nucleolus are associated with transcriptional repression in both in vivo-derived and in vitro-cultured oocytes in the presence of an intact cumulus oophorus (de la Fuente et al. 2001 Dev. Biol. 229, 224). It is widely accepted that oocyte communication with the somatic cell compartment is essential for both oocyte growth and acquisition of meiotic competence (Eppig et al. 1997 Hum. Reprod. 12, 127). In particular, cumulus cells play an active role in modulating the levels of transcription in the nucleoplasm and in perinuclear domains as well as in chromatin configuration of GV stage oocytes. In cattle, a heterogeneous population of cumulus-oocyte complexes (COCs) has been found after isolation from the follicle, and this is characterized by a different functional degree of gap junction-mediated communication (Luciano et al. 2004 Biol. Reprod. 70, 465). This study was aimed at investigating the possible correlation between the chromatin configuration of immature bovine oocytes and the status of communication between the oocyte and cumulus cells, and oocyte developmental competence. In the first experiment, 138 COCs, isolated from follicles 2–6 mm in diameter, were injected with a 3% solution of Lucifer Yellow to assess the communication status between oocytes and cumulus cells. Successively, COCs were freed of cells, and denuded oocytes (DOs) were stained with Hoechst 33342 to determine the chromatin configuration. In a second experiment, 330 COCs were denuded and stained with Hoechst 33342 in order to assess chromatin configuration and then matured in vitro according to their GV stage. After IVM, DOs were fertilized, and presumptive zygotes were cultured for 7 days at which time blastocyst rate was assessed. Data were analyzed by ANOVA and Fisher's PLSD test. Three stages of GV oocytes were identified: GVI, with filamentous chromatin distributed in the nucleoplasm; GVII, with chromatin condensed into thick clumps; and GVIII, with chromatin condensed into a single clump. The GVIII stage showed a lower proportion of functional open communication than the GVI and GVII groups (8.5 vs. 45.7 and 46.1, respectively, P < 0.05). However, when compared with each other, the GVI stage oocytes showed lower embryonic developmental competence (12.9 in GVI vs. 22.1 and 24.2 in GVII and GVIII, respectively, P < 0.05). Our findings indicate that the status of communication between oocytes and cumulus cells could be related to the chromatin organization in immature bovine oocytes. A direct correlation between the communications grade, the modulation of oocyte transcriptional activity, and the acquisition of oocyte developmental competence remain to be confirmed. This work was supported by a 2003 UniMi Grant.

65 OVULATION OF IMMATURE OOCYTES WITH HIGH COMPETENCE RATES

2017 ◽  
Vol 29 (1) ◽  
pp. 140
Author(s):  
A. M. Taiyeb ◽  
S. A. Muhsen-Alanssari ◽  
M. E. Kjelland ◽  
S. M. Taiyeb ◽  
A. I. Haji ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Control Group ◽  
Cortical Granule ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes ◽  
Antral Follicles ◽  
Significant Difference ◽  
High Competence

Collection of immature oocytes from antral follicles in superovulated mice is a widely established technique for retrieval of germinal vesicle (GV) and metaphase I (MI) oocytes. Investigators use their experience to select antral follicles under a microscope before puncturing the follicles. This is sometimes followed by screening of oocytes based on morphology and diameter, and usually mouse oocytes of small diameters or abnormal morphologies are excluded. Shortcomings with the current technique may include varied oocyte yields and collection of oocytes from primary and secondary follicles. Moreover, such immature oocytes were observed to have different chromatin configurations, cortical granule distributions, spindle-chromosome organizations, fertilization rates, and diameters. This study was designed to investigate the potential of ovulated immature oocytes, resultant from superovulated mice treated with an FDA approved phosphodiesterase 3A inhibitor named cilostazol (CLZ), to substitute for ovarian immature oocytes collected from antral follicles of superovulated mice. Swiss Webster mice were superovulated and gavaged with 7.5 mg of CLZ once, at the same time as hCG injection, or twice, at the same time as hCG plus 6 h post-hCG injection, to result in ovulation of MI or GV oocytes, respectively. Control ovarian GV or MI oocytes were collected from ovarian antral follicles of superovulated mice not treated with CLZ. Ten mice were used in each treatment or control group. Single or multiple administrations of CLZ resulted in mice ovulating 85.8 ± 3.9% MI oocytes or 95.2 ± 3% GV oocytes (mean ± SEM), respectively. Treated GV oocytes had significantly higher rates of advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had lower cAMP levels and higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P < 0.0001). Treated MI oocytes had significantly higher rates of normal spindle and chromosomes aligned at the metaphase plates and offspring than did control MI oocytes. Control or treated GV oocytes were found to have greater diameters than did control or treated MI oocytes, respectively (P < 0.007), indicating that initiation of meiotic maturation is associated with reduction in oocyte diameters and utilisation of cytoplasm proteins and cofactors. Moreover, control GV oocytes were found to have greater diameters than did treated GV oocytes (P = 0.007). This may refer to the readiness of treated GV oocytes to undergo germinal vesicle breakdown and transition into the MI stage, especially treated GV oocytes had high rates of meiotic development in comparison to control GV oocytes. Diameters of GV nuclei in treated GV oocytes were smaller than those in control GV oocytes (P = 0.006), which may also indicate a germinal vesicle that had started to undergo germinal vesicle breakdown. A similar significant difference was also noted with control and treated MI oocytes. In summary, we present a novel method for retrieval of immature oocytes at different stages of meiotic maturation. Treated ovulated immature oocytes had more uniform diameters and high developmental competence than did ovarian immature oocytes. Treated ovulated immature oocytes may substitute for ovarian immature oocytes and become an additional research resource.

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