Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

2018 ◽  
Vol 30 (10) ◽  
pp. 1298 ◽  
Author(s):  
Jianwei Huang ◽  
YongShun Ma ◽  
Shao Wei ◽  
Bo Pan ◽  
Yu Qi ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Oocyte Vitrification ◽  
Kegg Pathway Database ◽  
Global Transcriptome ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
Regulation Of Actin Cytoskeleton ◽  
Mrna Transcriptome

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2 h and 24 h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n = 3; vitrified GV, n = 4; fresh MII, n = 1 and MII derived from vitrified GV, n = 2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function–gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.

scholarly journals 321EFFECTS OF BUTYROLACTONE-I AND CYCLOHEXIMIDE ON GERMINAL VESICLE BREAKDOWN IN BOVINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT AFTER IVM-IVF-IVC

2004 ◽  
Vol 16 (2) ◽  
pp. 280
Author(s):  
M. Narita ◽  
I. Kei ◽  
O. Dochi
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Lower Percentage ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Sperm Penetration ◽  
Vitro Development ◽  
Bovine Oocytes

The present study aimed to compare the effects of butyrolactone-I (BL-I) and cycloheximide (CHX) on inhibition of germinal vesicle (GV) breakdown (GVDB) in bovine oocytes and subsequent in vitro development after in vitro maturation and fertilization. Furthermore, in experiment 2, we compared the kind of supplemented protein with CHX during inhibition of GVBD of oocytes obtained from ovaries stored for 1 day, and examined time extension of storage of oocytes. In experiment 1, bovine cumulus-oocyte complexes (COCs) collected by the aspiration of 3- to 5-mm follicles of ovaries from at a local abattoir were preincubated for 24h in TCM-199 supplemented with 100μM BL-I and 3mgmL−1 BSA or 100μLmL−1 CHX and 5% CS. As a control, fresh COCs were used without preincubation. In experiment 2, the COCs were collected from ovaries stored in phygiological saline for 1 day at 20°C. The collected COCs were preincubated for 24h in TCM-199 supplemented with 100μLmL−1 CHX and 3mgmL−1 BSA or 5% CS (CHX+BSA, CHX+CS). As a control, fresh COCs collected from ovaries stored in the same condition were used without preincubation. In both experiments, the COCs were maturated and inseminated with frozen-thawed spermatozoa. After preincubation, maturation and fertilization, some oocytes or zygotes were fixed to assess the rates of oocytes at the GV stage, MII or sperm penetration. Following insemination, the presumptive zygotes were cultured in CR1aa (Rosenkrans, C.F. Jr. et al., 1993 Biol. Reprod. 49, 459–462) supplemented with 5% CS for 8 days. Embryo development was evaluated for cleavage rates on Day 2, and for blastocyst rates on Days 7 and 8 (IVF=Day 0), respectively. To evaluate embryo quality, the total cell numbers in the blastocysts were counted by means of the air-drying method. Three replicates were carried out for each experiment. Data were analyzed by chi-square test (cleavage and blastocyst rates) and ANOVA (cell numbers). In experiment 1, there were no differences in the rates of the oocytes at the GV stage between BL-I (71.4±10.7%, mean±SD) and CHX (86.7±10.9%), but the rates of the oocytes at the MII stage for BL-I (59.6±7.4%) tended to be lower than for those in CHX (80.0±14.1%, P<0.1). The rate of MII stage for control was 67.5±18.4%, and there were no differences between control and other treatments. No differences were found in sperm penetration, normal fertilization and polyspermy after in vitro fertilization. The cleavage rate for oocytes in CHX (81.0±1.2%) was significantly higher than for those in BL-I (65.0±13.1%, P<0.01), and there was a tendency for the cleavage rate in BL-I to be lower than that of the control (75.5±4.7%, P<0.1). A significantly lower percentage of embryos cultured in BL-I (19.2±13.8%) developed to the blastocyst stage than those of embryos in the control (32.0±11.2%, P<0.05), but there were no differences in the blastocyst rate between BL-I and CHX (25.9±8.8%). Cell numbers in the blastocysts in BL-I (177.2±15.9, n=21) and CHX (191.2±12.9, n=31) were not significantly different compared to the control (198.4±14.3, n=34). In experiment 2, no significant differences were found in the cleavage rates (CHX+CS, 64.0±18.7%; CHX+BSA, 68.1±10.8% and control, 72.2±8.3%). However, the blastocyst rates in CHX+CS (4.0±7.8%) and CHX+BSA (7.7±9.2%) were significantly lower than the control (20.4±3.7%, P<0.05). These results suggested that CHX can reversibly inhibit the GVBD of bovine oocytes for 24h without compromising subsequent developmental competence after in vitro maturation, fertilization, and culture. However, COCs collected from stored ovaries for 1 day and preincubated with CHX failed to develop into blastocysts regardless of the kind of supplemented protein.

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.

Methodological approaches for vitrification of bovine oocytes

Zygote ◽  
2020 ◽  
pp. 1-11
Author(s):  
Linda Dujíčková ◽  
Alexander V. Makarevich ◽  
Lucia Olexiková ◽  
Elena Kubovičová ◽  
František Strejček
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Oocyte Vitrification ◽  
Factors Affecting ◽  
Oocyte Stage ◽  
Follicle Size ◽  
Oocyte Donors ◽  
Methodological Approaches ◽  
Bovine Oocytes

Summary Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Before And After ◽  
Vitro Development ◽  
Bovine Oocytes

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.

114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 183
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Dna Hydroxymethylation ◽  
Oocyte Collection ◽  
Grade 3 ◽  
Bovine Oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles &gt;2mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means±standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P&lt;0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88±2.34 and 14.23±2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25±3.69 and 27.59±3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80±2.44 and 22.60±2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82±4.54 and 55.87±3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92±4.04 and 91.11±3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90±71,793.86 and 313,819.88±55,528.01, respectively) and DNA hydroxymethylation (444,931.10±67,920.78 and 352,254.68±56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36±14,449.47 and 89,807.26±11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83±15,517.70 and 137,622.45±11,826.86 AU, respectively) of MII-stage oocytes.

scholarly journals Effect of concentration and exposure period to butyrolactone I on meiosis progression in bovine oocytes

2006 ◽  
Vol 58 (3) ◽  
pp. 354-359 ◽  
Author(s):  
P.R. Adona ◽  
C.L.V. Leal
Keyword(s):  
Drug Concentration ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Exposure Period ◽  
Culture Period ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Kinetics Of

The effect of concentration and exposure period of bovine oocytes to butyrolactone I (BLI) on meiotic block and in vitro maturation (IVM) kinetics was studied. In experiment 1, all oocytes were at germinal vesicle stage (GV), after 6h in culture with 0, 50 and 100µM BLI. After 12h, all oocytes cultured with 50 and 100µM BLI remained in GV. After 24h, less oocytes were in GV with 50µM (82%) than with 100µM BLI (99%, P<0.05). In experiment 2, after 6h IVM, 93% of control oocytes (IVM only) were in GV, while treated oocytes (100µM BLI for 6, 12 or 24h prior to IVM) showed less oocytes in GV with increased exposure period to BLI prior to IVM (83 and 73%, for 6h and 12h, P<0.05). For a 24h inhibition, GV rates were similar to 12h (70%, P>0.05). After 18h IVM, metaphase II (MII) rates were similar for all groups (76-81%). In experiment 3, after 6h IVM, 74% of treated oocytes (50 or 100µM BLI for 12h) were in GV. This rate was lower than for control oocytes (97.3%, P<0.05). After 18h IVM more oocytes (~80%, P>0.05) were in MII with BLI than for control (73%, P<0.05). Shorter culture periods require lower BLI concentration for meiotic block; initial nuclear maturation kinetics of oocytes cultured with BLI is accelerated, and this is affected by culture period but not by drug concentration.

scholarly journals The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†

2020 ◽  
Vol 103 (5) ◽  
pp. 1000-1011 ◽  
Author(s):  
Katrin Gegenfurtner ◽  
Florian Flenkenthaler ◽  
Thomas Fröhlich ◽  
Eckhard Wolf ◽  
Georg J Arnold
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Substantial Effect ◽  
Developmental Competence ◽  
High Rate ◽  
Transcription Inhibition ◽  
The Impact ◽  
Bovine Oocytes

Abstract Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC–MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell–cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

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