Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

2008 ◽  
Vol 103 (3-4) ◽  
pp. 336-347 ◽  
Author(s):  
H.S. Lee ◽  
X.J. Yin ◽  
Y.X. Jin ◽  
N.H. Kim ◽  
S.G. Cho ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Chromatin Configuration ◽  
Meiotic Competence

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 (<1, 1–2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <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<0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <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.

scholarly journals The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes

Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 639-643 ◽  
Author(s):  
Michele Bellone ◽  
Maurizio Zuccotti ◽  
Carlo Alberto Redi ◽  
Silvia Garagna
Keyword(s):  
Germinal Vesicle ◽  
Chromatin Organization ◽  
Developmental Competence ◽  
Cell Stage ◽  
Morphological Marker ◽  
Good Marker ◽  
Chromatin Configuration ◽  
Meiotic Competence

Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus (SN, chromatin forms a ring around the nucleolus), and not surrounded nucleolus (NSN, chromatin has a diffuse pattern). Oocytes of both classes are capable of meiotic resumption, but while SN oocytes, following fertilization, develop to term, NSN oocytes never develop beyond the two-cell stage. A recent study has shown that the position of the germinal vesicle (GV) can be used as a morphological marker predictive of oocyte meiotic competence, i.e. oocytes with a central GV have a higher meiotic competence than oocytes with an eccentric GV. In the present study, we have associated both markers with the aim of identifying, with more accuracy, the oocytes' developmental competence. Following their isolation, antral oocytes were classified on the basis of both SN and NSN chromatin configuration and their GV position, matured to metaphase II and fertilized in vitro. We demonstrated that the position of the GV is a good marker to predict the oocytes' developmental competence, but only when associated with the observation of the chromatin organization.

scholarly journals Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth

Zygote ◽  
1995 ◽  
Vol 3 (4) ◽  
pp. 325-332 ◽  
Author(s):  
Yuji Hirao ◽  
Youki Tsuji ◽  
Takashi Miyano ◽  
Akira Okano ◽  
Masashi Miyake ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Early Growth ◽  
Catalytic Subunit ◽  
The Other ◽  
Antral Follicles ◽  
Stages Of Growth ◽  
Mean Diameter ◽  
M Phase ◽  
Chromatin Configuration ◽  
Meiotic Competence

SummaryThe molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 μm, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 μm, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3–4 mm) and large antral follicles (5–6 mm) had mean oocyte diameters of 115 and 119 μm respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 μm) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 μm) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

scholarly journals Oocyte Meiotic Competence in the Domestic Cat Model: Novel Roles for Nuclear Proteins BRD2 and NPM1

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniela R. Chavez ◽  
Pei-Chih Lee ◽  
Pierre Comizzoli
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Domestic Cat ◽  
Nuclear Proteins ◽  
Protein Levels ◽  
Nuclear Factors ◽  
Chromatin Configuration ◽  
Mammalian Female ◽  
Adult Cat ◽  
Meiotic Competence

To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.

198 CILOSTAMIDE SUSTAINS GAP JUNCTION-MEDIATED COMMUNICATION AND CHROMATIN REMODELLING IN PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
C. Dieci ◽  
F. Franciosi ◽  
V. Lodde ◽  
I. Lagutina ◽  
I. Tessaro ◽  
...  
Keyword(s):  
Gap Junction ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Period ◽  
Control Group ◽  
Functional Coupling ◽  
Mediated Communication ◽  
Developmental Potential ◽  
Chromatin Configuration ◽  
Defined Culture

In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P < 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P < 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).

Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop

2013 ◽  
Vol 25 (8) ◽  
pp. 1204 ◽  
Author(s):  
Adel R. Moawad ◽  
Jie Zhu ◽  
Inchul Choi ◽  
Dasari Amarnath ◽  
Wenchao Chen ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Control Groups ◽  
Blastocyst Development ◽  
Chromosome Configuration ◽  
Nuclear Maturation ◽  
Chromatin Configuration ◽  
And Control ◽  
First Time

The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus–oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P < 0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

2003 ◽  
Vol 15 (4) ◽  
pp. 223 ◽  
Author(s):  
Muhammad Rizwan Yousaf ◽  
Kazim Raza Chohan
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Morphology ◽  
Vitro Fertilization ◽  
Size Dependent ◽  
Size Groups ◽  
Follicle Size ◽  
Fetal Bovine ◽  
Meiotic Competence

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL−1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL−1 follicle-stimulating hormone, 10 μg mL−1 luteinizing hormone, 1.5 μg mL−1 oestradiol, 75 μg mL−1 streptomycin, 100 IU mL−1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.

311 TEMPERATURE DURING OVERNIGHT HOLDING IN MEIOSIS INHIBITOR-FREE MEDIUM AFFECTS CHROMATIN CONFIGURATION AND MEIOTIC RESUMPTION IN EQUINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 244
Author(s):  
N. A. Martino ◽  
M. E. Dell'Aquila ◽  
M. F. Uranio ◽  
R. Lampignano ◽  
G. M. Lacalandra ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Chi Squared ◽  
Chromatin Configuration ◽  
Higher Temperature ◽  
Meiotic Inhibitors ◽  
Multiple Comparison Methods ◽  
And Control

Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955–963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142–1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P < 0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P < 0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P < 0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n = 29) and control (n = 36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2′,7′-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720–728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8 ± 499.5 v. 722.9 ± 390.3; DCF fluorescence intensity, 278.5 ± 179.3 v. 378 ± 185, and mt/ROS colocalization, 0.5 ± 0.1 v. 0.5 ± 0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.

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