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.

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.

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

Differential accumulation of oocyte nuclear proteins by embryonic nuclei of Xenopus

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 829-846 ◽  
Author(s):  
C. Dreyer
Keyword(s):  
De Novo ◽  
Germinal Vesicle ◽  
Intrinsic Property ◽  
Nuclear Proteins ◽  
Cell Nuclei ◽  
Germinal Vesicle Breakdown ◽  
Early Stages ◽  
Nuclear Antigens ◽  
Gradual Process ◽  
Oocyte Nuclear Proteins

Oocyte nuclear proteins of Xenopus are distributed into the cytoplasm of the maturing egg after germinal vesicle breakdown. Later they are found in all cell nuclei of the embryo. At early stages of development, different nuclear proteins behave differently. A class of ‘early shifting’ antigens is accumulated by pronuclei and cleavage nuclei, whereas others appear to be excluded from the nuclei at early stages but are shifted into the nuclei at blastula or during and after gastrulation. Accumulation of ‘late-shifting’ nuclear antigens is a gradual process and occurs during a period characteristic of each protein. Multiple artificial pronuclei can be formed after injection of sperm nuclei, erythrocyte nuclei or pure lambda-DNA into unfertilized eggs. The artificial pronuclei accumulate early- but not late-shifting proteins. Early-migrating proteins rapidly accumulate into the germinal vesicle after de novo synthesis in the oocyte, indicating that the efficiency of translocation into nuclei is an intrinsic property of each protein. Artificial extension of the length of the cell cycle before midblastula transition does not lead to accumulation of the late-shifting nuclear antigens investigated.

Effects of slow freezing and vitrification on embryo development in domestic cat

2020 ◽  
Vol 55 (10) ◽  
pp. 1328-1336
Author(s):  
Valentina I. Mokrousova ◽  
Konstantin A. Okotrub ◽  
Eugeny Y. Brusentsev ◽  
Elena A. Kizilova ◽  
Nikolai V. Surovtsev ◽  
...  
Keyword(s):  
Embryo Development ◽  
Domestic Cat ◽  
Slow Freezing

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).

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

45 Expression patterns of PRDM family genes in porcine pre-implantation embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 148
Author(s):  
K. Farrell ◽  
K. Uh ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Internal Control ◽  
Target Genes ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Cell Stage ◽  
Early Embryo Development

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values&lt;0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.

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