39 Efficacy of roscovitine and dibutyryl cAMP to block premature meiosis in porcine oocytes vitrified at the germinal vesicle stage and their effect on subsequent embryo development

2022 ◽  
Vol 34 (2) ◽  
pp. 254
Author(s):  
N. Hiep ◽  
T. Somfai ◽  
Y. Hirao ◽  
T. Dang-Nguyen ◽  
N. Men ◽  
...  
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Dibutyryl Camp

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.

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 (&lt;1, 1–2, &gt;2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles &lt;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&lt;0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles &lt;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 effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage

2015 ◽  
Vol 15 (2) ◽  
pp. 115-120 ◽  
Author(s):  
Hassan Pazoki ◽  
Hussein Eimani ◽  
Farah Farokhi ◽  
Abdol-Hossein Shahverdi ◽  
Leila Sadat Tahaei
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Platelet Lysate ◽  
Nmri Mouse ◽  
Mouse Oocytes

scholarly journals 226HEAT SHOCK OF GERMINAL VESICLE-STAGE BOVINE OOCYTES REDUCES EMBRYO DEVELOPMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 234 ◽  
Author(s):  
R.R. Payton ◽  
A.M. Saxton ◽  
J.L. Lawrence ◽  
J.L. Edwards
Keyword(s):  
Elevated Temperature ◽  
Heat Shock ◽  
Embryo Development ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Cell Stage ◽  
Experimental Control ◽  
Maturation Medium ◽  
Linear Contrast ◽  
Humidified Air

Culture of germinal vesicle (GV)-stage oocytes at an elevated temperature occurring in heat-stressed dairy cattle reduced ability of oocytes to progress to metaphase II after resumption of meiosis (Payton RR et al., 2003 Biol. Reprod. 68, 343 abst). The objective of this study was to evaluate embryo development of oocytes heat-shocked at GV stage. To prevent cumulus-oocyte complexes from resuming meiosis after removal from follicles, oocytes were cultured in roscovitine (cell cycle inhibitor of p34cdc2/cyclin B kinase;; 50μM) for 24h (McCann LM et al., 2000 Biol. Reprod. 64, 141 abst and Payton RR et al., 2003 Biol. Reprod. 68, 343 abst) showed that roscovitine is effective for maintaining &gt;90% of oocytes at GV-stage in a reversible manner. Germinal vesicle-stage oocytes were cultured at 38.5°C for 24h (experimental control) or 41.0°C as follows: HS 0–6 (41°C for 6h, 38.5°C for 18h), HS 0–12 (41°C for 12h, 38.5°C for 12h), HS 12–24 (38.5°C for 12h, 41°C for 12h), HS 18–24 (38.5°C for 18h, 41°C for 6h), or HS 0–24 (41°C for 24h) in 5.5% CO2 and humidified air. In addition, a group of COC were not cultured in roscovitine but placed in maturation medium (lab control). After a total of 24h, COC were washed extensively of roscovitine and cultured for an additional 24h in maturation medium. Oocytes presumed mature were fertilized with Percoll-prepared frozen-thawed semen. Presumptive zygotes were cultured in KSOM containing 1X nonessential amino acids in 5.5% CO2, 7% O2, and 87.5% N2 at 38.5°C in humidified air. Cleavage and development to blastocyst were recorded on Days 3 and 8 post-insemination, respectively. Data were collected in 7 replicates and analyzed as an incomplete block using mixed models of SAS (2000) after testing for normality. Use of roscovitine for maintaining oocytes at GV-stage for 24h did not alter cleavage (80.5 and 73.4%; SEM=5.8; lab and experimental controls), development to 8–16 cell (50.4 and 52.6%; SEM=4.6; lab and experimental controls), or blastocyst (29.7 and 24.8%; SEM=3.2; lab and experimental controls) stages. Culture of GV-stage oocytes at 41°C for up to 24h did not increase lysis (8.0–11.1%; SEM=2.7). Heat shock of GV-stage oocytes for as few as 6h reduced the proportion developing to 8–16 cell stage after release from inhibitor (Table 1). When experimental control and HS 0–6 were pooled for comparison to HS 0–12, effects of heat shock for reducing development to blastocyst were noted (P&lt;0.005). Moreover, negative effects of heat shock for reducing developmental competence of GV-stage oocytes increased as duration of heat shock increased (linear contrast;; experimental control, HS 0–12, and HS 0–24; P&lt;0.04). Results indicate that a physiologically relevant elevated temperature for as few as 6h compromises continued development of GV-stage oocytes. Seasonal depressions in fertility of heat-stressed cattle may be due in part to direct effects of elevated temperature on GV-stage oocytes. Table 1

Mouse oocytes penetrated by sperm at GV or GVBD stage lose the ablity to fuse with additional spermatozoa

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 123-128 ◽  
Author(s):  
Beata Pyrzyńska ◽  
M. Maleszewski ◽  
D. Maluchnik
Keyword(s):  
Plasma Membrane ◽  
Meiotic Division ◽  
Germinal Vesicle ◽  
Cortical Granule ◽  
Dibutyryl Camp ◽  
Granule Exocytosis ◽  
Mouse Oocytes ◽  
Sperm Nuclei ◽  
Cortical Granule Exocytosis

SummaryMouse oocytes penetrated by spermatozoa during germinal vesicle (GV) breakdown undergo maturation and are arrested at metaphase of the second meiotic division despite the presence of sperm nuclei within the ooplasm. When these oocytes were re-inseminated, none was penetrated by newly added spermatozoa. When GV oocytes were inseminated and cultured in the presence of dibutyryl cAMP, the oocytes remained at GV stage, yet they did not permit entry of additional spermatozoa. These observations suggest that the plasma membrane of maturing oocytes is modified by precociously penetrating spermatozoa independently from cortical granule exocytosis. Sperm components incorporated into the oocytes seem to be responsible for the modification of the oocyte's plasma membrane.

scholarly journals Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

2007 ◽  
Vol 22 (7) ◽  
pp. 1982-1990 ◽  
Author(s):  
B. Heindryckx ◽  
P. De Sutter ◽  
J. Gerris ◽  
M. Dhont ◽  
J. Van der Elst
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle

scholarly journals Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

2020 ◽  
pp. 1
Author(s):  
Tulay Irez ◽  
Sinem Ercan Dogan ◽  
Enver Ciraci ◽  
Saadet Busra Aksoyer ◽  
Muhammet Sait Toprak ◽  
...  
Keyword(s):  
Experimental Study ◽  
Luteinizing Hormone ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Immature Oocytes ◽  
Maturation Rate

<p><strong>OBJECTIVE:</strong> In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. </p><p><strong>STUDY DESIGN:</strong> In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. </p><p><strong>RESULTS:</strong> In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).</p><p><strong>CONCLUSION:</strong> In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.</p>

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.

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