scholarly journals Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants

2018 ◽  
Vol 48 (6) ◽  
Author(s):  
Taís da Silva Lopes ◽  
Eduardo Antônio Sanches ◽  
Danilo Caneppele ◽  
Elizabeth Romagosa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Propylene Glycol ◽  
Germinal Vesicle ◽  
Blue Staining ◽  
Mature Stage ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes ◽  
Stage Of Development ◽  
Oocyte Stage

ABSTRACT: To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter <1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.

scholarly journals Aspects of cytoplasmic maturation of bovine oocytes: Interplay between mapk, mRNA-cap binding complex and cytoplasmic mRNA metabolism in regulation of translation

2003 ◽  
Vol 19 (3-4) ◽  
pp. 1-8 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Melo Sterza ◽  
M. Kubelka ◽  
Z. Vohnikova ◽  
W. Tomek
Keyword(s):  
Binding Protein ◽  
Germinal Vesicle ◽  
Initiation Factor ◽  
Meiotic Maturation ◽  
Detailed Knowledge ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Stage Of Development ◽  
Bovine Oocytes

Bovine oocytes are arrested in the germinal vesicle stage (GV stage)and mature spontaneously when they are removed from their follicles and transferred to a suitable culture medium. This process, known as meiotic maturation is characterized among others, by germinal vesicle breakdown followed by metaphase I (MI) stage and further development to metaphase II (MII), where they become arrested again. During GVBD to MI transition, the overall protein synthesis reaches the highest level and it rapidly declines in MII. We have previously shown that transcription completely declines during meiotic maturation. Therefore we suppose that gene expression is exclusively regulated on translational level at this stage of development. This means that mRNAs, which were stored in repressed form during oocyte growth, were actively translated during meiotic maturation. Therefore we have investigated specific regulators of translation, namely the eukaryotic initiation factor of translation eIF4E (cap binding protein) and a specific repressor of eIF4E function, the 4E-binding protein 4E-BP1. Furthermore, we have elucidated pathways, which lead to eIF4E and 4E-BP1 phosphorylation by using specific M-phase kinase inhibitors, and we compare these results with transcription and cytoplasmic polyadenylation events during the course of meiotic maturation. The detailed knowledge of such regulatory processes can help to improve in vitro bio-techniques and to estimate the risk of these techniques.

274 LIPOLYSIS IN CUMULUS CELLS ACCOMPANIES OOCYTE MATURATION IN BOVINE

2015 ◽  
Vol 27 (1) ◽  
pp. 226 ◽  
Author(s):  
S. Uzbekova ◽  
L. Sanchez-Lazo ◽  
A. Desmachais ◽  
V. Maillard ◽  
S. Elis
Keyword(s):  
Oocyte Maturation ◽  
Energy Homeostasis ◽  
Binding Proteins ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hormone Sensitive Lipase ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

Oocyte maturation relies on energy from different nutrients, including fatty acids (FA). Cumulus cells (CC) are metabolically coupled with enclosed oocyte and active FA metabolism occurs in both compartments. Excess of lipids in oocyte environment alters its developmental competence. Lipid droplets (LD), mainly composed of triacylglycerides (TG), are formed inside of CC and in oocyte to store lipids. Liberation of free FA from TG requires lipolysis, which is catalyzed by lipases and involves FA-binding proteins (FABP) and perilipins (PLIN), which interact at the surface of LD as shown in lipogenic tissues. The objective was to elucidate the main factors involved in lipolysis in bovine cumulus-oocyte complex (COC) during oocyte maturation. Gene expression before and after maturation was analysed in CC by microarray hybridization and validated by real time RT-PCR; proteins were detected by Western blot and immunofluorescence. For statistics, ANOVA and Mann-Whitney (M-W) tests were used. In CC, adipose triglyceride lipase PNPLA2, lipoprotein lipase LPL, and monoacylglycerol lipase ABHD6 showed the highest mRNA expression level among 7 detected lipases. Both PLIN5 and PLIN2 were the most abundant perilipins, and among 8 FA-binding proteins, FABP3 and FABP5 were predominant. During in vitro maturation (IVM), expression of most of these genes increased at 6 h of IVM (P < 0.05, ANOVA) in CC. At that time, germinal vesicle breakdown occurred in enclosed oocytes and hyaluronan synthase HAS2, involved in the extra-cellular matrix formation, was upregulated in CC. The most upregulated genes after 18 h of IVM in CC were ABDH6 (48.5-fold as compared to immature, P < 0.01, M-W), FABP3 (16.6-fold, P < 0.01, M-W), and PLIN2 (5.5-fold, P < 0.05, M-W). Expression of all of these lipolysis-related genes was also detected in the oocytes. At the protein level, PLIN2 was mainly localised in the cytoplasmic LD, both in CC and in the oocyte. In CC, FABP3 was detected in the cytoplasm, whereas in oocyte it was also localised to the germinal vesicle of immature oocytes and closely to the chromosomes during the first meiotic division. In addition, active phosphorylated hormone sensitive lipase HSL was always detected in CC and in mature oocytes, but not in immature oocytes. All these data demonstrate that lipolysis occurs both in CC and in the oocyte during maturation. Lipolysis may be necessary to maintain cell energy homeostasis by regulating intracellular concentration of free FA. Moreover, CC were already described to store the excess FA from follicular fluid in order to protect the oocyte. Our data corroborate the essential role of CC in oocyte survival through controlling FA metabolism inside the COC. Active lipolysis may therefore be required to reduce lipid storages as well as to produce energy necessary for oocyte meiosis progression and extracellular matrix secretion by CC in order to prepare COC for further fertilization.This work was supported by INRA, ANR (OSCILE project) and European subvention FP7-KBBE-2012–6 (FECUND project).

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Lun Suo ◽  
Guang-Bin Zhou ◽  
Qing-Gang Meng ◽  
Chang-Liang Yan ◽  
Zhi-Qiang Fan ◽  
...  
Keyword(s):  
Survival Rate ◽  
Cell Damage ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

SummaryCryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Germinal Vesicle ◽  
Storage Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Storage Solutions ◽  
Stock Solutions ◽  
Phosphate Buffered Saline

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.

scholarly journals In vitro development of mouse fetal germ cells into mature oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Wei Shen ◽  
Lan Li ◽  
Zhaodai Bai ◽  
Qingjie Pan ◽  
Mingxiao Ding ◽  
...  
Keyword(s):  
Genomic Imprinting ◽  
Germ Cells ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Primordial Follicles ◽  
Ovarian Cells ◽  
Fetal Mice ◽  
Vitro Development

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish anin vitroexperimental model that allows one to study such mechanisms. Mouse follicular development has been studiedin vitroover the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were culturedin vitrofor 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were maturedin vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula–blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normallyin vitro.

scholarly journals Effects of gonadotrophin in vivo and 2-hydroxyoestradiol-17β in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis

2006 ◽  
Vol 189 (2) ◽  
pp. 341-353 ◽  
Author(s):  
A Mishra ◽  
K P Joy
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Hplc Method ◽  
Heteropneustes Fossilis ◽  
Germinal Vesicle Breakdown ◽  
Steroid Profile ◽  
17 Hydroxyprogesterone ◽  
Envelope Layer

An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20β-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 μM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20β-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 μM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20β-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20β-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.

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