Comparison of maturational and developmental parameters of oocytes recovered from prepubertal and adult pigs

2003 ◽  
Vol 15 (4) ◽  
pp. 215 ◽  
Author(s):  
Koji Ikeda ◽  
Yoshiyuki Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Rate Of Development ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Before And After ◽  
Maturation Rate

To clarify the cause(s) of the differences between the developmental competence of prepubertal and adult porcine oocytes, the following were examined: (i) the meiotic progression, p34cdc2 kinase activity, ooplasm diameter and response to activation stimuli of the oocytes; and (ii) the development of parthenotes and nuclear transfer (NT) embryos obtained using oocytes recovered from prepubertal and adult pigs. Oocytes were recovered from 4- to 8-mm follicles of abattoir-derived ovaries. There were no apparent differences in the morphology of the germinal vesicle, nuclear maturation rate, activity of p34cdc2 kinase or response to parthenogenetic stimulation between prepubertal and adult oocytes. Before and after maturation culture, the ooplasm diameters of prepubertal oocytes were smaller than those of adult oocytes. Parthenotes and NT embryos derived from prepubertal gilt oocytes showed a lower rate of development to the blastocyst stage than those derived from adult oocytes. These results suggest that lower developmental competence of prepubertal oocytes may be caused by their inability to complete ooplasmic maturation, and that this is not because of altered oocyte maturation kinetics and/or p34cdc2 kinase activity. Furthermore, the smaller diameter of the ooplasm of prepubertal oocytes indicates that most oocytes that have routinely recovered from prepubertal gilt ovarian follicles are still in the growing phase and have immature ooplasm.

Investigations of oocyte in vitro maturation within a mouse model

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Boon Chin Alexis Heng ◽  
Ng Soon Chye
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Standard Duration ◽  
Maturation Rate ◽  
Meiotic Competence

This study attempted to develop a ‘less meiotically competent’ murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze–thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

Effects of vitrification of cumulus-enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion, nuclear progression and cytoplasmic maturation

2017 ◽  
Vol 29 (12) ◽  
pp. 2419 ◽  
Author(s):  
Ruth Appeltant ◽  
Tamás Somfai ◽  
Elisa C. S. Santos ◽  
Thanh Quang Dang-Nguyen ◽  
Takashi Nagai ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Nuclear Morphology ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Oocyte Aging ◽  
Cytoplasmic Maturation ◽  
And Control

Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus–oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.

scholarly journals Transcriptome Analyses Reveal Differential Transcriptional Profiles in Early- and Late-Dividing Porcine Somatic Cell Nuclear Transfer Embryos

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1499
Author(s):  
Zhiguo Liu ◽  
Guangming Xiang ◽  
Kui Xu ◽  
Jingjing Che ◽  
Changjiang Xu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Rna Processing ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Molecular Mechanisms ◽  
Differentially Expressed Gene ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Rna Seq ◽  
Transcriptional Profiles

Somatic cell nuclear transfer (SCNT) is not only a valuable tool for understanding nuclear reprogramming, but it also facilitates the generation of genetically modified animals. However, the development of SCNT embryos has remained an uncontrollable process. It was reported that the SCNT embryos that complete the first cell division sooner are more likely to develop to the blastocyst stage, suggesting their better developmental competence. Therefore, to better understand the underlying molecular mechanisms, RNA-seq of pig SCNT embryos that were early-dividing (24 h postactivation) and late-dividing (36 h postactivation) was performed. Our analysis revealed that early- and late-dividing embryos have distinct RNA profiles, and, in all, 3077 genes were differentially expressed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that early-dividing embryos exhibited higher expression in genes that participated in the meiotic cell cycle, while enrichment of RNA processing- and translation-related genes was found in late-dividing embryos. There are also fewer somatic memory genes such as FLRT2, ADAMTS1, and FOXR1, which are abnormally activated or suppressed in early-dividing cloned embryos. These results show that early-dividing SCNT embryos have different transcriptional profiles than late-dividing embryos. Early division of SCNT embryos may be associated with their better reprogramming capacity, and somatic memory genes may act as a reprogramming barrier in pig SCNT reprogramming.

scholarly journals Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

2021 ◽  
Vol 9 ◽  
Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam
Keyword(s):  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Current Approach ◽  
Developmental Competence ◽  
Matrix Remodeling ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

32 MicroRNA-29b Improves the Quality and Developmental Potential of Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 155
Author(s):  
W.-J. Zhou ◽  
S. Liang ◽  
X.-S. Cui
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Down Regulation ◽  
Developmental Potential ◽  
Somatic Cell Reprogramming ◽  
Underlying Mechanisms

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse cellular processes. miR-29b plays a crucial role during somatic cell reprogramming. However, studies of the function of miR-29b in embryogenesis are limited. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with IVF embryos (P < 0.05). To determine the function of miR-29b in the bovine SCNT embryo, we microinjected a miR-29b mimic and inhibitor into bovine SCNT zygotes. The results showed that miR-29b significantly decreased the expression of Dnmts (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos (P < 0.05). We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency (P > 0.05) but down-regulation inhibits developmental potency (P < 0.05). Although miR-29b overexpression does not improve the developmental potency of bovine SCNT embryos, the quality of bovine SCNT embryos at the blastocyst stage improved significantly (P < 0.05). The expression of pluripotency factors (OCT4 and SOX2) and cellular proliferation rate were significantly higher in blastocysts from the miR-29b overexpression group than the control and down-regulation groups (P < 0.05). In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and down-regulation groups (P < 0.05). Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

191 Effect of the time of oocyte collection through slicing method on meiosis resumption and invitro embryo production

2020 ◽  
Vol 32 (2) ◽  
pp. 224
Author(s):  
S. Soto-Heras ◽  
A. Lorenzo ◽  
I. Menéndez-Blanco ◽  
D. Izquierdo ◽  
M. Paramio
Keyword(s):  
Acetic Acid Solution ◽  
Germinal Vesicle ◽  
Random Variable ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Collection ◽  
Group 3

Oocytes from juvenile goats are collected by slicing the ovary surface because the high percentage of small antral follicles limits follicular aspiration. The time of oocyte collection can impair oocyte developmental competence due to spontaneous resumption of meiosis. The aim of this study was to assess whether the time of slicing period affects oocyte meiosis and embryo development after invitro fertilization. Ovaries from juvenile goats (1-2 months old) were recovered at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected by slicing, selected, and kept in the slicing medium at 38.5°C in humidified air with 5% CO2 until analysis or culture. The slicing medium was HEPES-buffered (25mM) TCM-199 with 2.2mgmL−1 NaHCO3 and 50mgmL−1 gentamicin. Two slicing periods were tested: T1 (1 h) and T4 (4 h). After this time, a group of oocytes were stained with 1% orcein in 45% acetic acid solution for assessing meiotic arrest and observed as the rate of germinal vesicle (GV; 61-67 oocytes/group from 5 replicates). The remaining COCs were cultured in our conventional IVM medium (TCM-199 with FSH, LH, oestradiol, sodium pyruvate, glutamine, cysteamine, epidermal growth factor, and fetal bovine serum) at 38.5°C with 5% CO2. After 24h, a sample of oocytes were stained for assessing nuclear maturation (28-29 oocytes/group, 3 replicates), and the rest were invitro fertilized with 4×106 spermmL−1 in BO-IVF medium (IVF Bioscience) for 20h and embryo cultured in BO-IVC medium for 7 days (70-81 oocytes/group, 3 replicates). Blastocysts were stained with Hoechst 33258 for determining the number of cells. Data were analysed with two-way ANOVA with RStudio version 1.2.1335. The time of slicing was set as a fixed factor and the replicate as random variable. Data presented as percentage did not follow a normal distribution and were square root arcsine transformed before analysis. At the end of slicing periods T1 and T4, oocytes at GV were 100% and 84.7±5.0%, respectively (P&lt;0.05). After 24h of IVM, the oocytes at MII were 77.0±7.1% and 88.6±7.3%, respectively, without statistical differences. However, oocytes from T1 produced a higher rate of cleaved oocytes (84.6±0.9%) and expanded blastocysts (11.03±5.2%) than T4 (49.8±7.9%, 0%, respectively; P&lt;0.05). The total blastocyst rate for T1 and T4 was 25.4±5.8% and 9.4±4.9%, respectively (P=0.068). No differences were observed in blastocyst cell number (75.9±4.0 and 67.5±10.9, respectively). In conclusion, oocytes resume meiosis before IVM during a long slicing period, even though the slicing medium is not supplemented with hormones or growth factors. The longer slicing period does not affect nuclear maturation but impairs oocyte competence, observed as lower cleavage and blastocyst development. Further experiments are needed to determine whether the use of meiotic inhibitors in the slicing medium can prevent the negative effect of the long slicing period. This study was funded by the Spanish Ministry of Science, Innovation and Universities (AGL2017-85837-R).

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.

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