scholarly journals In Vitro Maturation of Fully Grown Mouse Antral Follicles in the Presence of 1 nM 2-Hydroxyestradiol Improves Oocytes’ Developmental Competence

2020 ◽  
Vol 28 (1) ◽  
pp. 121-133
Author(s):  
Valeria Merico ◽  
Mario Zanoni ◽  
Alexis Parada-Bustamante ◽  
Silvia Garagna ◽  
Maurizio Zuccotti
Keyword(s):  
Germinal Vesicle ◽  
Asymmetric Division ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Antral Follicles ◽  
Blastocyst Quality ◽  
Quality Markers ◽  
Estradiol Metabolites

AbstractCathecolestrogens are estradiol metabolites produced during folliculogenesis in the mammalian ovary. 2-Hydroxyestradiol (2-OHE2) is one of the most abundant although its role remains unknown. The aim of this study is to investigate whether the presence of 2-OHE2 during the germinal vesicle-to-metaphase II transition affects oocyte meiotic and preimplantation developmental competence. Mouse cumulus-oocyte complexes (COCs), isolated from fully grown antral follicles, were in vitro–matured (IVM) in the presence of 2-OHE2 (0.1, 1, 10 or 100 nM) for 6 or 15 h; then, their meiotic and developmental competence was evaluated using a number of cytological quality markers. With the exception of the highest dose (100 nM), the addition of 2-OHE2 to the IVM medium, did not alter, compared with untreated control, the frequency of oocytes that reached the MII stage. Instead, IVM in the presence of 1 nM 2-OHE2 highly increased the rate of preimplantation development and blastocyst quality. To understand whether this positive effect could be attributed to the events occurring during meiosis resumption, we analysed a number of specific cytological quality markers of the asymmetric division, such as PB-I volume and position, presence and extension of the cortical F-actin cap, meiotic spindle shape and area, and microtubule organisation centre localisation. The results highlighted how the presence of 1 nM 2-OHE2 significantly improved the overall cytological organisation required for a correct asymmetric division. Our results contribute a first step to acknowledge a potential role of this estradiol metabolite during the GV-to-MII transition, contributing to the acquisition of oocytes developmental competence.

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 759-769 ◽  
Author(s):  
T Tharasanit ◽  
S Colleoni ◽  
G Lazzari ◽  
B Colenbrander ◽  
C Galli ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Germ Line ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Oocyte Cryopreservation ◽  
Meiotic Spindle ◽  
Maturation Stage ◽  
Blastocyst Formation ◽  
Female Germ Line

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte’s stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified–warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Germinal Vesicle ◽  
Storage Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Storage Solutions ◽  
Stock Solutions ◽  
Phosphate Buffered Saline

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 °C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 °C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Effect of season on germinal vesicle stage, quality, and subsequent in vitro developmental competence in bovine cumulus-oocyte complexes

2021 ◽  
pp. 103171
Author(s):  
Francisco Báez ◽  
Ramiro López Darriulat ◽  
Nélida Rodríguez-Osorio ◽  
Carolina Viñoles
Keyword(s):  
Germinal Vesicle ◽  
Developmental Competence

scholarly journals Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells

2002 ◽  
Vol 172 (1) ◽  
pp. 45-59 ◽  
Author(s):  
F Le Bellego ◽  
C Pisselet ◽  
C Huet ◽  
P Monget ◽  
D Monniaux
Keyword(s):  
Estrous Cycle ◽  
Granulosa Cells ◽  
Physiological Role ◽  
Follicular Development ◽  
Antral Follicles ◽  
Final Development ◽  
Beta 1 Integrin ◽  
Arginine Glycine Aspartic Acid

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro

1997 ◽  
Vol 9 (6) ◽  
pp. 603 ◽  
Author(s):  
J. C. Bell ◽  
L. C. Smith ◽  
R. Rumpf ◽  
A. K. Goff
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Uv Exposure ◽  
One Dimensional ◽  
Repair Mechanisms ◽  
Bovine Oocytes

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulusoocyte complexes (COCs) were denuded before or after maturationin vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high-possibly due to nuclear repair mechanisms. Protein proles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specic proteins are synthesized in the nuclear region.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

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