Large-scale chromatin remodeling in germinal vesicle bovine oocytes: Interplay with gap junction functionality and developmental competence

2007 ◽  
Vol 74 (6) ◽  
pp. 740-749 ◽  
Author(s):  
Valentina Lodde ◽  
Silvia Modina ◽  
Cristina Galbusera ◽  
Federica Franciosi ◽  
Alberto M. Luciano
Keyword(s):  
Gap Junction ◽  
Chromatin Remodeling ◽  
Large Scale ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Bovine Oocytes

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.

236 NUCLEAR AND CYTOPLASMIC MODIFICATIONS OF GERMINAL VESICLE BOVINE OOCYTES IN RELATION TO CHROMATIN REMODELING

2006 ◽  
Vol 18 (2) ◽  
pp. 226
Author(s):  
V. Lodde ◽  
P. Maddox-Hyttel ◽  
S. Modina ◽  
A. M. Luciano
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Structural Modifications ◽  
Chromatin Configuration ◽  
Hoechst Staining ◽  
Bovine Oocytes

We previously reported that germinal vesicle (GV) bovine oocytes can be classified on the basis of their chromatin organization and that increased chromatin condensation is accompanied by a major incidence of gap junction-mediated coupling interruption between germ and cumulus cells and by an increase in oocyte developmental competence (Lodde et al. 2005 Reprod. Fertil. Dev. 17(2), 294-295). The aim of this study was to characterize, at the ultrastructural level, both nuclear and cytoplasmic compartments of bovine oocytes classified according to their chromatin configuration because key structural modifications, such as nucleolar inactivation and remodeling of specific ooplasmic structures, take place during the later phases of oocyte growth. Cumulus-oocyte complexes collected from 0.5-2-mm early antral (EA) and 2-6-mm mid-antral (MA) follicles were freed of cumulus cells. Denuded oocytes were stained with Hoechst 33342, classified according to the degree of chromatin condensation, and processed for light microscopy of semi-thin sections (LM; n = 10 in each class) and transmission electron microscopy (TEM; n = 5 in each class). Four classes of oocytes were identified by the Hoechst staining: GV0 with filamentous chromatin diffused in the nuclear area, GV1 with few foci of condensed chromatin, GV2 with chromatin further condensed into distinct clumps, and GV3 with chromatin condensed into a single clump. Almost all oocytes collected from EA follicles were classified as GV0. Oocytes of this class were absent in MA follicles, whereas class GV1, GV2, and GV3 oocytes occurred at similar frequency. LM confirmed the chromatin condensation found by the Hoechst staining and revealed that in class GV2 and GV3 oocytes the chromatin was mainly located close to the nucleolus. Ultrastructurally, the nucleolus was fibrillo-granular in GV0 oocytes; the oocytes in the other classes displayed an electron dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most organelles were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex, were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Our findings indicate that the process of chromatin remodeling is strictly related to structural modifications that characterize the later stages of the oocyte growth phase. Because the highest degree of chromatin condensation was combined with degenerative features of cortical granules, we hypothesize that this class of oocytes (GV3) originated from early atretic follicles, as also suggested in other species. The evaluation of oocytes on the basis of chromatin configuration may be useful for the development of new strategies for manipulating fertility in mammals. This work was supported by a COFIN Grant.

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.

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.

scholarly journals Role of Gap Junction-Mediated Communications as Regulators of Large-Scale Chromatin Remodeling During Final Differentiation of Bovine Oocyte.

2009 ◽  
Vol 81 (Suppl_1) ◽  
pp. 281-281
Author(s):  
Valentina Lodde ◽  
Federica Franciosi ◽  
Silvia Modina ◽  
Irene Tessaro ◽  
Alberto M. Luciano
Keyword(s):  
Gap Junction ◽  
Chromatin Remodeling ◽  
Large Scale ◽  
Bovine Oocyte

scholarly journals 241 EFFECT OF MEIOTIC ARREST BY ROSCOVITINE AND SUBSEQUENT IVM TIME ON DEVELOPMENTAL COMPETENCE OF IMMATURE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 271
Author(s):  
L. Campos-Chillon ◽  
T. Suh ◽  
E. Carnevale ◽  
G. Seidel
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Sperm Concentration ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Group ◽  
Meiotic Arrest ◽  
Cell Stage ◽  
Bovine Oocytes

Maintaining immature bovine oocytes at the germinal vesicle stage by inhibiting M-phase promoting factor (MPF) activity is a reversible process when using roscovitine, and this can improve cytoplasmic maturation in vitro. However, optimum meiotic arrest times and subsequent IVM times have not been determined, so we evaluated the developmental competence of oocytes in relation to these times. Two by two factorial treatments consisting of 2 arrest times (8 h, 16 h) and 2 subsequent IVM times (16 h, 22 h) plus a control were replicated 6 times in this study. Semen from two bulls was used three times. Chemically defined media (CDM) were used throughout (Olson and Seidel 2000 J. Anim. Sci. 78, 152–157). Slaughterhouse-derived oocytes were arrested in meiosis in 1 mL of CDM-M without any hormones, but containing 50 μM roscovitine and 0.5% fatty acid-free (FAF)-BSA under 5% CO2 in air at 38.5°C. After 8 or 16 h of meiotic arrest, oocytes were washed and matured in 1 mL of CDM-M containing 0.5% FAF-BSA, 2 mM glucose, 50 ng/mL EGF, 15 ng/mL NIDDK-oFSH-20, 1 μg/mL USDA-LH-B-5, 1 μg/mL E2, and 0.1 mM cysteamine for 16 or 22 h under 5% CO2 in air at 38.5°C. Oocytes for the control group were matured in 1 mL of the CDM-M with hormones for 22 h. Ten oocytes from each group were fixed after IVM, stained with orcein, and evaluated for maturation to MII. For fertilization, motile sperm recovered from frozen-thawed semen were co-incubated for 18–20 h with ∼20 oocytes/group at a final sperm concentration of 0.5 × 106 sperm/mL in F-CDM. Presumptive zygotes were cultured in 0.5 mL of CDM-1 for 2.5 days and then in CDM-2 for 5.5 days in 5% CO2, 5% O2, 90% N2 in a humidified incubator at 39°C. Cleavage rates were evaluated after culture in CDM-1. Blastocyst rate, blastocyst stage (5 = early, 6 = full, 6.5 = expanding, 7 = expanded, 7.5 = hatching, 8 = hatched), and embryo quality (1 = excellent, 2 = good, 3 = fair, 4 = poor) were evaluated after CDM-2. Data were subjected to ANOVA; the arc sin transformation was used for percentage data, and least-squares means are presented. There were no significant differences in % cleavage (Cle), cell stage, or blastocyst quality among treatments (P > 0.1). However, meiotic arrest of oocytes for 16 h and subsequent IVM for 16 h improved embryo development to blastocysts compared to other roscovitine treatments (Table 1, P < 0.05). A bull effect for % blastocysts was observed, 19.9% and 25.2% for bulls 1 and 2, respectively (P < 0.08). Blastocyst production was improved by shortening oocyte maturation time from 22 to 16 h, when meiotic progression was previously inhibited for 16 h with roscovitine. Table 1. Effect of meiotic arrest and IVM times on oocyte maturation and embryo development

scholarly journals The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†

2020 ◽  
Vol 103 (5) ◽  
pp. 1000-1011 ◽  
Author(s):  
Katrin Gegenfurtner ◽  
Florian Flenkenthaler ◽  
Thomas Fröhlich ◽  
Eckhard Wolf ◽  
Georg J Arnold
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Substantial Effect ◽  
Developmental Competence ◽  
High Rate ◽  
Transcription Inhibition ◽  
The Impact ◽  
Bovine Oocytes

Abstract Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC–MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell–cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Developmental competence of gametes reconstructed by germinal vesicle transplantation from fresh and cryopreserved bovine oocytes

2010 ◽  
Vol 93 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Federica Franciosi ◽  
Federica Perazzoli ◽  
Valentina Lodde ◽  
Silvia C. Modina ◽  
Alberto M. Luciano
Keyword(s):  
Germinal Vesicle ◽  
Developmental Competence ◽  
Bovine Oocytes
Sign in / Sign up

Export Citation Format

Share Document