Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Shogo Higaki ◽  
Masao Kishi ◽  
Keisuke Koyama ◽  
Masashi Nagano ◽  
Seiji Katagiri ◽  
...  

SummaryThe preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

2014 ◽  
Vol 26 (6) ◽  
pp. 806 ◽  
Author(s):  
Yong-Xun Jin ◽  
Ming-Hui Zhao ◽  
Zhong Zheng ◽  
Jung-Suk Kwon ◽  
Seul-Ki Lee ◽  
...  

Previous studies show that porcine oocyte aging resulting from asynchronised IVM impairs embryo developmental competence. In the present study we investigated whether trichostatin A (TSA; an inhibitor of histone deacetylation) prolongs the maturation time and prevents the aging of oocytes. Porcine oocytes were cultured in medium containing increasing concentrations of TSA (300 nM) for 24, 44 or 64 h. The percentage of oocytes that underwent germinal vesicle breakdown was significantly lower in the TSA-treated group (300 nM) than in the control group. TSA did not affect oocyte quality at MII based on levels of maturation-promoting factor, the phosphorylation status of mitogen-activated protein kinase or histone H3K9 acetylation analysis. We also compared the preimplantation developmental competence and the viability of pathenogenetic embryos treated with 100 nM TSA for 24 h and then continuously cultured for another 24 h in TSA free condition. No significant differences were observed for either parameter between the TSA-treated and control groups. These results indicate that TSA prolongs the IVM of porcine oocytes but that oocyte quality and aging are not affected. These findings provide a feasible option by which to adjust the initiation time of downstream experiments based on porcine matured oocytes.


2007 ◽  
Vol 19 (8) ◽  
pp. 947 ◽  
Author(s):  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
W. M. Chis Maxwell ◽  
Simon K. Walker

Two experiments were conducted in Merino lambs to examine the effects of gonadotrophin-releasing hormone (GnRH) treatment on the developmental competence of oocytes collected after pretreatment with follicle stimulating hormone (FSH). The first experiment examined the effects of six GnRH treatment times (control and GnRH administered 2, 4, 6, 8 and 10 h before oocyte collection) and four in vitro maturation (IVM) periods (18, 20, 22, 24 h) on the rate of oocyte nuclear maturation. The second experiment examined the effect of five GnRH treatment times (control and GnRH administered 2, 4, 6 and 8 h before oocyte collection) and three IVM periods (20, 22, 24 h) on the development of oocytes and embryos after in vitro maturation, fertilisation and culture. In Experiment 1, GnRH treatment did not influence the mean number of cumulus-oocyte-complexes (COCs) collected or COC morphology at the time of collection. However, treatment changed (P < 0.01) the distribution of follicle size and this was primarily due to a marked reduction in the number of follicles with diameters <2 mm. In addition, GnRH treatment at 6 and 8 h increased (P < 0.01) the proportion of oocytes that developed to Metaphase II (MII) (63.2 and 72.6%, respectively) compared with other treatment times (range 52.9–59.9%). Nuclear maturation was influenced by a significant (P < 0.05) interaction between GnRH treatment and IVM period due to a disproportionately greater number of oocytes at the germinal vesicle breakdown (GVBD) stage for the 2 and 4 h GnRH treatments compared with other treatments. In Experiment 2, cleavage rate (range 63.5–85.9%) was highest when GnRH was administered 8 h before collection but the percentage of cleaved oocytes that developed into blastocysts (range 10.0–35.0%) was significantly (P < 0.05) lower for the 6 and 8 h GnRH treatments compared with the control and the 2 h GnRH treatment. These results demonstrate that GnRH treatment before oocyte collection can improve nuclear maturation and cleavage rates in lamb oocytes but that these improvements are not reflected in improved rates of blastocyst development. It is speculated that this discrepancy may result from GnRH treatment either adversely affecting cytoplasmic maturation or inducing asynchrony between the maturation of the nuclear and cytoplasmic components of the oocyte.


Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  

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.


Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.


Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.


2006 ◽  
Vol 18 (2) ◽  
pp. 279 ◽  
Author(s):  
L. Tubman ◽  
A. Peter ◽  
R. Krisher

Diphenyleneiodonium (DPI), an inhibitor of the pentose phosphate pathway (PPP), arrests nuclear maturation of porcine oocytes. This inhibition is reversed using products or cofactors of PPP such as nicotinamide adenine dinucleotide phosphate (NADP), phosphoribose diphosphate (PRPP), and ribose-5-phosphate (R5P). The objective of this study was to determine the relationship between DPI-mediated meiotic inhibition, reversal of this inhibition, and metabolism of in vitro-matured (IVM) porcine oocytes. Oocytes were aspirated, searched, and selected in the presence of DPI, with the exception of control oocytes. Oocytes were then matured in one of five treatments for 40 h in 7% CO2 in air at 39°C in defined Purdue Porcine Medium for maturation (PPMmat). Treatments included control, 50 nM DPI (DPI), DPI + 5 mM NADP (NADP), DPI + 12.5 mM PRPP (PRPP), and DPI + 10 mM R5P (R5P). Following IVM, oocytes were denuded by vortexing. Glycolysis and PPP activities were measured in 4 μL hanging drops containing labeled glucose (0.0125 mM 5-3H glucose and 0.482 mM 1-14C glucose, respectively) for 3 h in 6% CO2. Oocytes were then individually fixed in a 3:2:1 solution of ethanol:acetic acid:chloroform and stained with aceto-orcein for determination of meiotic stage (germinal vesicle = 1 through metaphase II = 7). Data were analyzed using one-way ANOVA. The use of DPI inhibited PPP and nuclear maturation; additionally glycolysis was decreased by DPI compared to control. Addition of NADP and PRPP increased both metabolic pathways and nuclear maturation compared to DPI. R5P restored glycolysis and nuclear maturation to control levels, and PPP to above the control level. There were no significant differences among meiotic stages relative to glycolytic activity. PPP activity was significantly different (values with different superscripts; P < 0.05) among oocytes of different meiotic stages (germinal vesicle = 0.24 ± 0.03ad, germinal vesicle breakdown = 0.40 ± 0.05bcde, condensed chromatin = 0.44 ± 0.05bcd, metaphase I = 0.45 ± 0.12abcd, anaphase = 0.76 ± 0.50abcde, telophase = 0.92 ± 0.17be, metaphase II = 0.74 ± 0.08be). Percentages of oocytes reaching MII were 43.48 (control), 2.08 (DPI), 28.30 (NADP), 18.18 (PRPP), and 46.94 (R5P). These results demonstrate that the PPP is a critical control mechanism for nuclear maturation of porcine oocytes, as inhibition of this metabolic pathway resulted in arrest of nuclear maturation. Addition of PPP cofactors or end products to the arresting medium led to reversal of inhibition as demonstrated by restoration of PPP activity resulting in nuclear maturation. Table 1. Meiotic stage, glycolysis, and pentose phosphate pathway activity after in vitro maturation of porcine oocytes


2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
M. Fathi ◽  
A. R. Moawad ◽  
M. R. Badr

Cryopreservation of oocyte would be an alternative to overcome the limited availability of dromedary camel oocytes and allow improvements in in vitro production in this species. Our aim was to develop a protocol for vitrification of dromedary camel oocytes at the germinal vesicle (GV) stage using various cryoprotectant combinations and cryo-carriers. In experiment 1, cumulus–ppcyte complexes (COC) obtained at slaughter were equilibrated in a solution composed of 10% ethylene glycol (EG) and 0.25 M trehalose. The oocytes were then exposed for 60 s to vitrification solutions (VS) composed of 20% EG and 20% dimethyl sulfoxide (DMSO; VS1) or 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3). The COC were then transferred into decreasing concentration of trehalose solution (toxicity test). In experiment 2, COC were randomly divided into 4 groups and vitrified by using straw or open pulled-straw (OPS) or solid surface vitrification (SSV) or cryotop in VS1 or VS2. Following vitrification and warming viable oocytes were matured in vitro for 30 h at 39°C in 5% CO2 in air. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels and then cultured in modified KSOMaa medium for 7 days. Oocyte viability, maturation, fertilization, and embryo development were evaluated. Data were analysed using one-way ANOVA and t-test. Viability and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%) than those exposed to VS1 (68.2% and 48.0%) and VS2 (79.3% and 56.9%). Although recovery rates were significantly lower (P ≤ 0.05) in oocytes vitrified using SSV or cryotop in either VS1 or VS2 solutions (66.9% to 71.1%) than those vitrified by straws using VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in SSV and cryotop groups (90.7% to 94.8%) than straw and OPS (68.2% to 86.5%) groups. Among vitrified groups, maturation and fertilization rates (51.8% and 39.2%, respectively) were the highest in the cryotop-VS2 group. Those values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and cryotop groups than in straws. No significant differences were observed in these parameters between cryotop and control groups. Together, the results show that both vitrification solution and cryodevice affect viability and developmental competence of vitrified/warmed dromedary camel oocytes. We report for the first time that dromedary camel oocytes vitrified at the GV stage have the ability to be matured, fertilized, and subsequently develop in vitro to produce blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.


2005 ◽  
Vol 17 (2) ◽  
pp. 3 ◽  
Author(s):  
Poul Maddox-Hyttel ◽  
Bolette Bjerregaard ◽  
Jozef Laurincik

The nucleolus is the site of rRNA and ribosome production. This organelle presents an active fibrillogranular ultrastructure in the oocyte during the growth of the gamete but, at the end of the growth phase, the nucleolus is transformed into an inactive remnant that is dissolved when meiosis is resumed at germinal vesicle breakdown. Upon meiosis, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities harbour the development of fibrillogranular nucleoli and re-establishment of nucleolar function in conjunction with the major activation of the embryonic genome. This so-called nucleologenesis occurs at a species-specific time of development and can be classified into two different models: one where nucleolus development occurs inside the NPBs (e.g. cattle) and one where the nucleolus is formed on the surface of the NPBs (e.g. pigs). A panel of nucleolar proteins with functions during rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) are localised to specific compartments of the oocyte nucleolus and those engaged in late processing are, to some degree, re-used for nucleologenesis in the embryo, whereas the others require de novo embryonic transcription in order to be allocated to the developing nucleolus. In the oocyte, inactivation of the nucleolus coincides with the acquisition of full meiotic competence, a parameter that may be of importance in relation to in vitro oocyte maturation. In embryo, nucleologenesis may be affected by technological manipulations: in vitro embryo production apparently has no impact on this process in cattle, whereas in the pig this technology results in impaired nucleologenesis. In cattle, reconstruction of embryos by nuclear transfer results in profound disturbances in nucleologenesis. In conclusion, the nucleolus is an organelle of great importance for the developmental competence of oocytes and embryos and may serve as a morphological marker for the completion of oocyte growth and normality of activation of the embryonic genome.


2021 ◽  
Vol 22 (20) ◽  
pp. 11148
Author(s):  
Radek Procházka ◽  
Alexandra Bartková ◽  
Lucie Němcová ◽  
Matej Murín ◽  
Ahmed Gad ◽  
...  

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.


2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
H. J. Oh ◽  
M. K. Kim ◽  
Y. H . Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  

In most mammals, maturation occurs within the ovarian follicle, and preovulatory oocytes are ovulated and ready for fertilization within the oviduct. In contrast, bitch ovulate primary oocytes, over a three day period, undergo both maturation and fertilization within the oviduct. The present study was conducted to evaluate the effects of canine synthetic oviduct fluid (cSOF) supplemented with the various energy substrates on in vitro maturation of canine oocytes. Oocytes were recovered by mincing ovaries collected after ovariohysterectomy in bitches at the follicular stage. Only oocytes with more than two layers of cumulus cells and with homogeneous cytoplasm >100 mm in diameter were selected. Then, oocytes cultured in tissue culture medium (TCM)-199 (control) or cSOF supplemented with various concentrations of glucose (0, 1.11, 3.89, or 5.56 mM, Exp. 1) or fructose (0, 1.11, 3.89, or 5.56 mM, Exp. 1), pyruvate (0, 0.1, 0.25, or 0.5 mM, Exp. 2) or lactate (0, 0.5, 1.0, or 5.0 mM, Exp. 3). In Exp. 4, the combined effects of glucose (1.11 mM), pyruvate (0.5 mM) and lactate (5.0 mM) on nuclear maturation of canine oocytes were investigated. A total of 2990 canine oocytes from 205 ovaries were used for experiments with replication at least three times. The oocytes were cultured for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. After 72 h, the oocytes were stained with 1.9 �g/mL Hoechst 33342 in glycerol and then evaluated under UV light to determine the stage of meiosis as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) with first polar body. The results of Exp. 1 showed that maturation of canine oocytes to MII was significantly higher (P < 0.05) in medium supplemented with 1.11 mM glucose (4.8%) than for the control (1.8%) and the other glucose-supplemented groups (0 to 1.8%). In Exp. 2, oocytes cultured in cSOF supplemented with 0.5 mM pyruvate showed a significantly higher (P < 0.05) maturation rate to MII (6.3%) than did the other pyruvate-supplemented (0, 0.8, or 2.5%) groups or the control (2.4%). In Exp. 3, more oocytes were matured to the MII stage in cSOF supplemented with 5.0 mM lactate (7.3%) than were the other lactate-supplemented groups (0 to 2.4%) or the control (2.5%). Results of Exp. 4 showed more oocytes progressed to MII in cSOF supplemented with 0.5 mM pyruvate (8.2%), 1.11 mM glucose + 0.5 mM pyruvate (7.4%), or 1.11 mM glucose + 0.5 mM pyruvate 0.5 + 5.0 mM lactate (7.3%) than did the other combination groups (2.2 to 5.2%). In conclusion, the present study demonstrated that supplementing cSOF with 1.11 mM glucose, 0.5 mM pyruvate, or 5.0 mM lactate significantly increased the maturation of canine oocytes to MII, and the combined supplementation of 1.11 mM glucose, 0.5 mM pyruvate, and 5.0 mM lactate further promoted oocyte nuclear maturation compared to 1.11 mM glucose alone and the control. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).


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