Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation

1999 ◽  
Vol 54 (4) ◽  
pp. 388-395 ◽  
Author(s):  
Bin Wu ◽  
Jiangen Tong ◽  
S.P. Leibo
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Meiotic Spindle ◽  
Spindle Formation ◽  
Bovine Oocytes

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.

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.

The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Zygote ◽  
2000 ◽  
Vol 8 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Seong Koo Cho ◽  
Seok Hwa Choi ◽  
Eun Young Kim ◽  
Se Pill Park ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Cytochalasin B ◽  
Germinal Vesicle ◽  
Condensed Chromatin ◽  
Meiotic Spindle ◽  
Cell Cortex ◽  
Germinal Vesicle Breakdown ◽  
Metaphase Stage ◽  
Bovine Oocytes

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.

250 CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN AND DYNACTIN p150Glued EXHIBIT DISTINCT SPATIAL AND TEMPORAL MICROTUBULE ASSOCIATIONS DURING BOVINE IN VITRO MATURATION AND ARE AFFECTED BY FOLLICLE SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
S. E. Racedo ◽  
M. C. Branzini ◽  
D. Salamone ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Intermediate Chain

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39�C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4�C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37�C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield�, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence.

168 EFFECT OF WORTMANNIN IN IN VITRO MATURATION OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 175
Author(s):  
E. M. Mogollón-Waltero ◽  
A. J. B. Dias ◽  
H. F. Gomes ◽  
D. F. Dubeibe ◽  
R. C. Maia ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Actin Filaments ◽  
In Vitro Maturation ◽  
Meiotic Spindle ◽  
Cortical Granules ◽  
Nuclear Maturation ◽  
Pi3k Activity ◽  
Blastocyst Rate ◽  
Bovine Oocytes

It has been shown that phosphatidylinositol 3 kinase (PI3K) participates in oocyte maturation by regulating the activity of important reactions related to the resumption of meiosis and energy metabolism. Changes in the enzyme activity caused by in vitro conditions may impair important events of oocyte maturation and consequently the production of blastocysts. The study aimed to verify the effect of the addition of wortmannin (a specific inhibitor of PI3K) to the in vitro maturation medium on nuclear and cytoplasmic maturation of bovine oocyte, as well as on the production of blastocysts. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with FCS and 0 (control) or 20 nM of wortmannin and then fertilized and cultured in vitro in the absence of inhibitor. Twenty-two hours after in vitro maturation the determination of PI3K activity of oocytes by Western blot was performed using anti-PI3K subunit P85 antibody/peroxidase. The activity quantification was performed by densitometry of the bands using the Gel Perfect software (Bozzo and Retamal 1991 Arch. Biol. Med. Exp. 63, 510). To check the effect of treatment on energy metabolism, glucose and glycogen concentration of oocytes was quantified by Glucox 500 test (Doles Reagentes e Equipamentos Para Laboratórios Ltd., Goiânia, Brazil). The oocyte viability determination was performed by double labelling with propidium iodide and calcein AM. To determine the nuclear maturation, the oocytes were stained with 2% acetic orcein, being considered matured those with chromosomes in metaphase plate. To assess the meiotic spindle organisation, the oocytes were labelled with anti-α tubulin-FITC and propidium iodide. The distribution of actin filaments and mitochondria was determined by rhodamine-phalloidin labelling. The distribution of cortical granules was observed by labelling the oocytes with Lens culinaris–fluorescein isothiocyanate (FITC). The cleavage and blastocyst rates were determined at 48 and 168 h post-fertilization, respectively, both calculated on the number of oocytes placed to mature. The treatment means were compared by t-test (SAS Institute Inc., Cary, NC, USA, 2003). Wortmannin produced a reduction around 40% of PI3K activity; however, the levels of glucose and glycogen were not altered. No changes were found in the viability of in vitro matured oocytes or in nuclear maturation rate (P ≥ 0.05). Treatment did not promote any change in the organisation of meiotic spindle, distribution of actin filaments, and positioning of mitochondria. However, oocytes treated with wortmannin showed a significant increase (P ≤ 0.05) in the migration of cortical granules compared with controls (87.4% ± 11.4 and 72.8 ± 11.8%, respectively). The cleavage rate was not influenced by treatment, but the blastocyst rate was higher when oocytes were matured in presence of wortmannin (34.2 ± 6.4% v. 20.0 ± 5.0%, respectively; P ≤ 0.05). The results indicate that partial inhibition of PI3K activity in bovine oocytes treated with wortmannin during in vitro maturation did not affect nuclear maturation, but improved the migration of cortical granules, which seems to have contributed to the increased the blastocyst rate.

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.

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