scholarly journals 253BOVINE OOCYTE CYCLIN B1 MRNA UNDERGOES CYTOPLASMIC POLYADENYLATION BEFORE THE BEGINNING OF IN VITRO MATURATION

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
K. Tremblay ◽  
C. Vigneault ◽  
G. Bujold ◽  
M.-A. Sirard
Keyword(s):  
In Vitro Maturation ◽  
De Novo ◽  
Mrna Level ◽  
Tail Length ◽  
Cyclin B1 ◽  
Cytoplasmic Polyadenylation ◽  
Cis Acting ◽  
Significant Difference ◽  
Bovine Oocytes

Maternal oocyte Cyclin B1 mRNA is known to be stored in the cytoplasm with a short poly(A) tail and be translationally dormant at GV stage. During maturation, Cyclin B1 poly(A) tail is elongated by a process called cytoplasmic polyadenylation and driven by A/U-rich cis-acting elements in its 3′ untranslated region (UTR) known as cytoplasmic polyadenylation elements (CPEs). The objective of this study was to elucidate whether GV-stage bovine oocytes possess a stockpile of Cyclin B1 mRNA stored with a short a poly(A) tail that is elongated during maturation by CPE regulation. The mRNA poly(A) tail length was measured by Rapid Amplification of cDNA Ends Polyadenylation test (Race-PAT) on oocytes (n=100) at the GV stage and 3, 5, 8, 10, 15, 20, and 25h of in vitro maturation. The mRNA poly(A) tail length was also measured in triplicate (n=20) on cold oocytes in GV (all manipulations on ice), warm oocytes in GV (ovaries transported in warm saline and manipulations on ice) and warm+2h 30min oocytes in GV (oocytes left for an additional 2h and 30min at room temperature). To assess for variation in mRNA quantity, Cyclin B1 mRNA level was quantified by real-time PCR (Lightcycler, Roche, Indianapolis, IN, USA) in cold, warm or warm+2h 30min GV oocytes (n=20). The data were treated as factorial design, using treatment and type of RT as factors, and analysed by ANOVA (SAS Inst., Cary, NC, USA). Differences between means were checked using Tukey’s test. Oocyte Cyclin B1 transcript show two different 3′ UTRs. These transcripts had the same ORF but different 3′ UTR lengths because of an alternative nuclear polyadenylation element AAUAAA (NPE). The longest form (Cyclin B1L) that possessed a putative CPE (UUUUAAUAAA) fused to the last NPE was studied. In warm GV oocytes, Cyclin B1L had a long poly(A) tail of 100 adenosine residues, and this length did not change during in vitro maturation. Interestingly, we found that Cyclin B1L showed an expected short poly(A) tail when the ovaries and the oocytes were transported and manipulated on ice. We showed that Cyclin B1L mRNA is cytoplasmically polyadenylated (addition of 75 adenosine residues) between the time of collection and the end of manipulation. This lengthening is most probably sufficient to promote translation. There was no significant difference between the Cyclin B1 mRNA quantity of cold oocytes or warm oocytes when the oligo used for the reverse transcription was either dt or decamers. Therefore, we believe that the increase in poly(A) tail length is not the result of Cyclin B1L mRNA degradation in cold oocytes or de novo transcription in warm oocytes. We report for the first time that Cyclin B1L cytoplasmic polyadenylation is carried out well before the beginning of in vitro maturation in bovine oocytes when ovaries are transported from the slaughterhouse in warm saline. Studying the real early mechanisms leading to resumption of meiosis in bovine oocytes is complicated by Cyclin B1 polyadenylation occurring prior to in vitro maturation. (Supported by NSERC.)

6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 121
Author(s):  
D. J. Walker ◽  
C. J. Wilusz ◽  
G. E. Seidel Jr
Keyword(s):  
In Vitro Maturation ◽  
Control Point ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Untranslated Regions ◽  
Labeled Rna ◽  
Bovine Oocytes

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

160 EFFECT OF cAMP MODULATORS ON IN VITRO MATURATION OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
S. E. Farmer ◽  
T. L. Adams ◽  
J. A. Sarmiento-Guzmán ◽  
C. L. Bailey ◽  
K. R. Bondioli
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Inhibitory Effect ◽  
Maturation Process ◽  
In Vitro Production ◽  
Angus Cattle ◽  
Significant Difference ◽  
Chi Squared ◽  
Bovine Oocytes

The efficiency of in vitro production (IVP) has remained low due to an inadequate in vitro maturation (IVM) system. Thus far, the most promising methods of improving IVM utilise cAMP modulators to slow the maturation process by keeping cAMP high. This experiment compares standard IVM to an extended IVM as previously described by Albuz (2010 Hum. Reprod. 25, 2999–3011). The extended IVM consists of a 2-h pre-IVM phase with FSK (an adenylate cyclase activator) and 3-isobutyl-1-methylxanthine [IBMX, a nonspecific phosphodiesterase (PDE) inhibitor], followed by a 31-h IVM phase with cilostamide (a PDE3 inhibitor). 3-Isobutyl-1-methylxanthine inhibits all PDE in both the oocyte and CC, whereas cilostamide inhibits PDE3 only in the oocyte, allowing a gradual reversal of inhibition. Bovine oocytes (n = 363) were obtained by transvaginal ultrasound-guided aspiration of both Brahman and Angus cattle over 4 collection days. Oocytes from each cow were divided into 2 groups. The first group was placed in standard 23-h IVM medium composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, penicillin and streptomycin, glutamine, and FSH, and cultured in 5% CO2 at 39°C. A subset of oocytes was removed from maturation at 8, 13, 18, and 23 h. The second group was placed into a pre-IVM medium of HEPES-TALP supplemented with 100 μM FSK and 500 μM IBMX for 2 h at 39°C, and then moved into standard maturation medium supplemented with 20 μM cilostamide for 31 h (5% CO2, 39°C). Oocytes were sampled at 8, 13, 18, 23, 28, and 33 h. Oocytes were stained with 1% orcein and nuclear status was examined for each sample time (Table 1). Data were analysed using a chi-squared test. At 8 h, there was a significant difference (P < 0.001) between GV stage of IVM and extended IVM (7.1 v. 73.2%), and between metaphase I (MI) stage IVM and extended IVM (76.2 v. 4.9%). At the 23-h time sample, there was a significant difference between metaphase II (MII) IVM and MII extended IVM (78.9 v. 30.6%; P < 0.001). There was also a significant difference between MII oocytes at 23 h IVM and MII oocytes at 33 h extended IVM (78.9 v. 33.3%; P < 0.001). These results are consistent with the hypothesis that cAMP modulators slow the nuclear maturation process. However, results also suggest that the inhibitory effect of the cAMP modulators is not completely reversible. Oocytes appear to arrest at MI by 23-h extended IVM and do not progress to MII at the same rate as standard IVM. Table 1.Nuclear status of bovine oocytes after standard IVM and extended IVM with cAMP modulators

172 ANTIVIRAL ACTION OF PROPOLIS AQUEOUS EXTRACT DURING MATURATION OF INFECTED BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 216
Author(s):  
E. G. Palazzi ◽  
D. Hansen ◽  
M. F. Alves ◽  
A. H. C. Nogueira ◽  
R. A. Ogata ◽  
...  
Keyword(s):  
Viral Replication ◽  
Aqueous Extract ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Main Step ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
Significant Difference ◽  
Bovine Oocytes

The objective of this study was to evaluate the decrease of virus replication in BoHV-1 (Colorado strain, 106.5 TCID50/mL) after the treatment using propolis aqueous extract (PAE) during in vitro maturation of infected bovine oocytes (24 h). Cow ovaries were obtained from a local slaughterhouse (Nelore breed) and transported to the laboratory. Cumulus-oocyte complexes (COC) were aspirated from follicles and separated into 4 groups (number of replicates for all groups = 6), which were exposed to 20 μL of sterile physiological solution (SPS), 100 μL of the in vitro maturation (IVM) medium [G1 (control), n = 609]; 10 μL of BoHV-1 (106.5 TCID50/mL) virus, 100 μL IVM medium, and 10 μL of SPS (G2, n = 786); 10 μL of PAE in 0.001% in SPS, 100 μL IVM medium, and 10 μL of SPS (G3, n = 819); 10 μL of PAE extract in 0.001% in SPS, 10 μL of BoHV-1 (106.5 TCID50/mL) virus and 100 μL of the IVM medium (G4, n = 734). All groups were kept for 24 h at 38.5°C, 5% CO2 in air. After the IVM, we analysed COC expansion and the presence of a polar body by optical microscope as well as viral replication by titration (Reed and Muench test) after 72 h of co-culture with Madin-Darby bovine kidney (MDBK) cells. The G1, G3, and G4 showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G2 did not have expansion of the cumulus cells. In contrast, the cytoplasm showed degenerative appearance and an absence of maturity in numerous oocytes. The maturation rates were as follows: G1 = 79% (482/609), G2 = 51% (407/786), G3 = 80% (662/819), and G4 = 76% (565/734). The differences among groups in maturation rates were compared using the chi-squared test (α = 5%) and the average titrations using the Mann–Whitney test (α = 5%). There was a significant difference (P < 0.01) among G1 and G2 evincing the interference of the virus maturation. The extract did not affect maturation as there was no difference among G1 and G3 (P = 0.43). The main step was finding no significant difference between the groups G1 and G4, (P = 17) proving that the extract interferes with viral replication. The titration after co-culturing the oocytes in MDBK demonstrated that G4 (average titrations = 1.63 × 103 titration) showed a lower rate of viral replication, the Mann-Whitney test, when compared to group G2 (average titrations = 6.04 × 107) which has not been subjected to treatment with PAE (P = 0.02). These results indicate that the propolis aqueous extract reduces the rate of viral replication without interfering with the maturation of oocytes and, therefore, it can be a conclusion that the analysis of the action of the molecules of this extract (by proteomics, for example) and future studies should be directed towards identifying the effect of extract on antiviral activity during the assessment of oocyte competence and embryo development.

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P &lt; 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P &lt; 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P &lt; 0.05) and its activity at 4 and 1 hpa, respectively (P &lt; 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P &lt; 0.05); however, its kinase activity decreased at 6 hpf (P &lt; 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P &lt; 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

Development to the late morula or blastocyst stage following in vitro maturation and fertilization of bovine oocytes

1989 ◽  
Vol 18 (1-3) ◽  
pp. 139-148 ◽  
Author(s):  
Y. Fukui ◽  
M. Urakawa ◽  
C. Sasaki ◽  
N. Chikamatsu ◽  
H. Ono
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Bovine Oocytes
Sign in / Sign up

Export Citation Format

Share Document