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BOR - Papers in Press, published online ahead of print December 15, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034793
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BIOLOGY OF REPRODUCTION 72, 1037–1044 (2005)
DOI: 10.1095/biolreprod.104.034793
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of Cyclin B1 Messenger RNA Isoforms and Initiation of Cytoplasmic Polyadenylation in the Bovine Oocyte1

Karine Tremblay, Christian Vigneault, Serge McGraw, and Marc-André Sirard2,

Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval,Sainte-Foy, Québec, Canada G1K 7P4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes can synthesize and store maternal mRNA in an inactive translational state until the start of in vitro maturation. Cytoplasmic polyadenylation, driven by 3'-untranslated region (UTR) cis-acting cytoplasmic polyadenylation element (CPE), is associated with translational activation of cyclin B1 mRNA during maturation. The main aim of the present study was to investigate if bovine oocyte cyclin B1 mRNA undergoes cytoplasmic polyadenylation/translation during in vitro maturation, as in other species. We have found that cyclin B1 mRNA is present in two isoforms, consisting of the same open reading frame but with different 3'-UTR lengths. Only the longest isoform (cyclin B1L) has a putative CPE sequence and other regulatory sequences, and its mRNA level decreases during early embryo development. The polyadenylation state of cyclin B1L during in vitro maturation was studied. Results demonstrated that cyclin B1L bears a relatively long poly(A) tail in germinal vesicle-stage oocytes, which is further lengthened at 10 h of maturation, before metaphase I. Interestingly, cyclin B1L bears a short poly(A) tail when the ovaries and the oocytes are transported and manipulated on ice to stop the polyadenylation process. Cytoplasmic polyadenylation most probably occurs during ovary transport in warm saline, when oocytes are still in their follicular environment. Our results also show a link between cytoplasmic polyadenylation of cyclin B1 and translation/appearance of cyclin B1 protein before in vitro maturation.

gamete biology, gene regulation, meiosis, ovum, ovum pick-up/ transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian ovary contains fully grown oocytes arrested at the dictyate stage of first meiotic prophase (for review, see [1]), also named the germinal vesicle (GV) stage. In vitro, resumption of meiosis occurs spontaneously on oocyte removal from its follicle (for review, see [2]), and one visible sign of meiosis resumption (maturation) is characterized by GV breakdown (GVBD). Resumption of meiosis is regulated by a complex called maturation-promoting factor (MPF), which is formed by association of the cyclin-dependent kinase (Cdk1 or p34cdc2) and cyclin B (for review, see [3, 4]), which can be either cyclin B1 or B2 in Xenopus oocytes (for review, see [5]). Activity of MPF is regulated by cyclin B binding to Cdk1 and the phosphorylation of three sites on Cdk1 (for review, see [6]).

Activity of MPF is low in GV-stage bovine oocytes and increases around GVBD to peak at the metaphase (M) I stage [7]. Many studies have shown the importance of bovine oocyte de novo protein synthesis for meiotic resumption [8–10]. One or multiple proteins implicated in meiotic resumption are synthesized during the first hours of in vitro culture (IVC) [8, 9], and cyclin B1 could be one of them [10]. Bovine oocyte cyclin B1 is absent in GV oocytes [10, 11] and is detected 3 h following the start of in vitro maturation (IVM) [10], 4 h before GVBD [12, 13]. The Cdk1 [7, 10, 11] and low levels of cyclin B2 [7] are present at the GV stage and, thus, may form pre-MPF. However, even if pre-MPF is present in bovine oocytes, protein synthesis is necessary for MPF activation [14] and GVBD [10, 14] (for review, see [15]). Interestingly, cyclin B1 protein microinjection is able to reverse oocyte GV arrest induced by cycloheximide, a protein synthesis inhibitor [10]. Therefore, initiation of cyclin B1 translation could be one of the early events leading to oocyte meiotic resumption.

Bovine GV-stage oocytes, recovered from slaughterhouse ovaries, possess a stockpile of maternal cyclin B1 mRNA [16] but no measurable protein [10]. Interestingly, it seems that cyclin B1 protein can accumulate before oocyte maturation, when the ovary transportation time is long [10]. In general, cyclin B1 mRNA probably is in a translationally inactive state (masked) in immature GV-stage bovine oocytes. Cytoplasmic polyadenylation has been described as a way of initiating translation of stored maternal mRNA during oocyte maturation and embryo development (for review, see [17]). Cytoplasmic polyadenylation during maturation requires two cis-acting 3'-untranslated region (UTR) sequences: the conserved hexanucleotide polyadenylation signal (HPS) AAUAAA, and the cytoplasmic polyadenylation element (CPE) sequence UUUUUA1–2U [18–23]. A general model of maternal mRNA masking (not translated) and unmasking (initiation of translation) was described and involves CPE-binding protein (CPEB)-maskin-eIF4E interactions (for review, see [24]). In Xenopus laevis oocytes, translational control of cyclin B1 during progesterone-induced oocyte maturation received great attention. In that species, the cyclin B1 3'-UTR bears cis-acting sequences (CPEs and others), which are responsible with trans-acting factors for translation repression in immature oocytes and initiation of translation (dependent on cytoplasmic polyadenylation) during oocyte maturation [21, 23, 25–30]. In mouse oocytes, endogenous cyclin B1 mRNA bears CPEs and is polyadenylated during maturation, reaching a peak at the MII stage [31]. Additionally, translational repression in immature oocytes and polyadenylation and translational activation in maturing oocytes is mediated by CPE in its 3'-UTR [31].

The first objective of the present study was to sequence and confirm cyclin B1 3'-UTR in GV-stage oocytes to identify further cis-acting elements that could regulate cyclin B1 mRNA polyadenylation/translation during maturation. The second objective was to depict steady-state mRNA level of cyclin B1 mRNA during maturation and embryo development to portray its depletion, which is characteristic of stored maternal mRNA. Our hypothesis, based on cyclin B1 3'-UTR analysis and cyclin B1 protein appearance, was that the mRNA is polyadenylated somewhere between ovary collection and a few hours following the start of IVM. To verify this hypothesis, cyclin B1 poly(A) tail was measured during maturation (GV stage and 3, 5, 8, 10, 15, 20, and 25 h of maturation) and also before maturation by comparing oocytes collected from ovaries transported in warm saline (normal procedure) or in cold saline (to stop the polyadenylation process). We also verified by real-time polymerase chain reaction (PCR) whether transcription of cyclin B1 mRNA occurred during ovary transport, a situation that could have biased our results regarding cytoplasmic polyadenylation. Finally, on the basis that conditions enabling cytoplasmic polyadenylation of cyclin B1 mRNA should result in translation initiation, we verified by Western blot analysis whether the protein was synthesized.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

RNA Extraction and Precipitation

All RNA extractions were done with the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA) as recommended by the manufacturer except for two subsequent 30-µl elutions with warm (60°C) elution buffer and an optional DNase treatment provided in the kit to remove genomic DNA. The RNA was precipitated by adding 6 µl of 3 M sodium acetate (pH 5.2), 1.5 µl of 1 mg/ml of linear acrylamide (Ambion, Austin, TX), and 105 µl of 100% isopropanol. The mixture was chilled at –80°C for 20 min and centrifuged at 15 000 rpm for 20 min. The pellets were washed once with cold 75% ethyl alcohol, centrifuged for an additional 5 min at 15 000 rpm, air-dried for 2–5 min, and resuspended in ddH2O.

3'-Rapid Amplification of cDNA Ends and Sequencingof Cyclin B1

To investigate if cyclin B1 mRNA could be found in different isoforms, 3'-rapid amplification of cDNA ends (RACE) and sequencing were performed. Denuded GV-stage oocytes (n = 400) were used to generate 3'-RACE cDNA with GeneRacer Kit version J (Invitrogen, Burlington, ON, Canada). The RACE cDNA synthesis was done as recommended by the manufacturer. The PCR amplification was done as follows: 1 µl of RACE cDNA, 1.5 µl of 10 µM cyclin B1 forward primer (5'-TCTTGTTGTTATGCAACACCTGGC-3'), 4.5 µl of 10 µM GeneRacer 3' reverse primer (5'-GCTGTCAACGATACGCTACGTAACG-3'), 5 µl of 10x PCR buffer, 1.5 µl of dNTP (10 mM of each; Qiagen, Mississauga, ON, Canada), and 0.5 µl of 5 U/µl of HotStar Taq DNA Polymerase (Qiagen), completed to a final volume of 50 µl with ddH2O. The cyclin B1 forward primer was designed based on bovine cyclin B1 sequence (GenBank accession no. L26548). The first PCR amplification conditions were as follows: 1 denaturation cycle of 15 min at 95°C, 34 PCR cycles (denaturation, 95°C for 1 min; annealing, 57°C for 1 min; extension, 72°C for 2 min), and 1 extension cycle for 7 min at 72°C. A nested PCR was done on the first PCR reaction with the following mix: 1 µl of the first PCR reaction, 1.5 µl of 10 µM cyclin B1 forward primer (5'-TCTTGTTGTTATGCAACACCTGGC-3'), 1.5 µl of 10 µM GeneRacer 3' nested reverse primer (5'-CGCTACGTAACGGCATGACAGTG-3'), 5 µl of 10x PCR buffer, 2 µl of dNTP (10 mM of each; Qiagen), and 0.5 µl of 5 U/µl of HotStar Taq DNA Polymerase (Qiagen), filled to 50 µl with ddH2O. The nested PCR amplification was done as described for the first PCR amplification. The PCR products were migrated on 2% agarose gel and the two expected bands cut out of the gel and purified with the QIAquick Gel Extraction Kit (Qiagen). Purified products were cloned into pCRII-TOPO with the TOPO TA Cloning Kit (Invitrogen). For each band, five clones were sequenced, and multiple alignments were analyzed [32] (see Fig. 1).



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  		FIG. 1. Alignment of cyclin B1S and cyclin B1L 3'-UTR and representation of cis-acting elements found in their 3'-UTR. Hexanucleotide polyadenylation signal (HPS), Sequence AATAAA in bold; CPE fused to the second HPS, sequence TTTTAATAAA underlined; putative class II ARE, sequence ATTTATTTA in bold and italicized. Note that cyclin B1S isoform lacks any CPE because of an alternate use of the first hexanucleotide for 3'-end processing

Experiment 1: Quantification of Long and Short Cyclin B1 Isoforms During Maturation and Development

Oocyte recovery and selection Ovaries were collected from slaughterhouse cows and transported to the laboratory in warm (initial temperature, 37–35°C; arrival temperature, 32–30°C) 0.9% NaCl aqueous solution containing antibiotic/antimycotic agents (saline). Aspiration and selection of cumulus-oocyte complexes (COCs) from follicles of 3–6 mm in diameter was performed as described by Vigneault et al. [33]. Pools of 40 denuded oocytes (triplicate) were frozen at –80°C after being denuded by vortexing and washed four times in PBS to remove cumulus cells.

IVM, in vitro fertilization, and IVC The COCs were washed and placed (n = 10) under mineral oil in 50-µl droplets of modified synthetic oviductal fluid (SOF) medium as described by Coenen et al. [13]. Maturation was conducted by incubation at 38.5°C with a 5% CO2 humidified atmosphere. The COCs were collected at 24 h (MII stage) of incubation. Denuded cumulus-free oocytes were frozen in groups of 40 (triplicate). A complete description of the in vitro fertilization and IVC has been provided previously [33]. Briefly, groups of 40 (triplicate) 2-cell embryos were collected and frozen at 36 h, 4-cell embryos at 48 h, and 8-cell embryos at 72 h postfertilization as well as blastocysts at 7 days of development.

cDNA synthesis for real-time PCR All pools of oocytes and embryos were spiked with 1 pg of exogenous GFP RNA (polyadenylated) in the initial lysis buffer before RNA extraction as described previously [33]. The long isoform of cyclin B1 (cyclin B1L) and the short isoform of cyclin B1 (cyclin B1S) were quantified in triplicate pools (n = 40) of GV-stage, MII-stage, and 2-, 4-, and 8-cell embryo and blastocyst cDNA prepared as described elsewhere [33].

Real-time PCR The cyclin B1L was amplified with primers specific for the transcript 3'-UTR (long up, 5'-GATCAGCACTCTAGCACAGC-3'; long low, 5'-AAAGGATAAGTAAAAAGAACTTCAAC-3') (see Fig. 2). Because the only difference between cyclin B1L and cyclin B1S is an extended 3'-UTR, we decided to amplify both transcripts (cyclin B1L+B1S; both up, 5'-ACCTGGCAAAGAATGTGGTC-3'; both low, 5'-GCTGTGCTAGAGTGCTGATCTTAG-3') with primers generating a common amplicon (see Fig. 2). A standard curve was made out of each PCR product, the specificity of which was verified by direct sequencing, purified with the QIAquick PCR Purification Kit (Qiagen), and quantified with a spectrophotometer. In each run, four standards were included, consisting of 1000, 100, 10, and 1 fg of PCR products. A Lightcycler apparatus (Roche Diagnostics, Laval, QC, Canada) was used for capillary amplification (SYBR green incorporation). The PCR reaction mixture consisted of the equivalent of the cDNA from a single oocyte or embryo, 0.5 µl of 10 µM of each primer, 1.6 µl of 25 mM MgCl2, 2 µl of SYBR green mix containing dNTPs, and FastStart DNA polymerase enzyme and buffer (Roche). Amplification conditions were as follows: 1 denaturation cycle of 10 min at 95°C, 50 PCR cycles (denaturation, 95°C for 1 sec; annealing, 59°C for 5 sec; extension, 72°C for 20 sec), a melting cycle from 70°C up to 95°C, and a final cooling cycle of 40°C for 30 sec. Lightcycler Software Version 3.5 (Roche) was used to quantify cDNA with comparison to the standard curve. Spiked exogenous green fluorescent protein (GFP) was quantified in each sample (all triplicates were done in the same run) in comparison to a GFP standard curve, and the quantity obtained was used to correct for loss of RNA during RNA extraction and precipitation and for reverse transcriptase and PCR efficiency. The highest GFP quantity was used to divide the GFP quantity obtained in each pool. The ratio obtained for each sample was then used to divide the quantity of products (cyclin B1L or cyclin B1L+B1S transcripts) obtained in the same sample. The products were verified by direct sequencing and migrated on agarose gel electrophoresis to confirm the presence of a single band product.



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  		FIG. 2. Alignment of cyclin B1S and cyclin B1L and schematic representation of primers used to quantify by real-time PCR of both cyclin isoforms (primers both up and both low) and the long isoform (primers long up and long low). Note that the sequences shown start at position 1237 of cyclin B1 mRNA (accession no. L26548). The stop codon is shown in bold and indicates the start of the 3'-UTR of both isoforms

Experiment 2: Measurement of Cyclin B1L Poly(A) Tail Length During Maturation

Ovaries collected from slaughterhouse cows were transported to the laboratory in warm (initial temperature, 37–35°C; arrival temperature, 32– 30°C) saline. Pools of 100 GV-denuded oocytes (duplicate) were frozen at –80°C after being denuded by vortexing and washed. The COCs were prepared and matured as described by Coenen et al. [13]. The COCs were collected at 3, 5, 8, 10, 15, 20, or 25 h of incubation. Denuded cumulus-free oocytes were frozen in groups of 100 (duplicate). Poly(A) tail length was evaluated by the polyadenylation test (RACE-PAT) as described below.

Experiment 3: Measurement of Cyclin B1L Poly(A) Tail Length and mRNA Quantification During Ovary Transport

Cold oocytes at time zero (C 0h) and warm oocytes at time zero (W 0h) were collected from slaughterhouse cow ovaries (follicle diameter, 3– 5 mm) and transported to the laboratory (4 h) either in ice-cold (C 0h) or warm (W 0h; initial temperature, 37°C) saline. Aspiration of COCs was done at room temperature for all treatments, but follicular fluid-containing COCs for treatment C 0h were put on ice. All COCs for treatment W 0h were manipulated (selected, denuded, and washed) at room temperature; in contrast, manipulations were done, as much as possible, in ice-cold conditions for treatment C 0h. Pools of 20 denuded oocytes were frozen. The polyadenylation test (RACE-PAT) was performed as described below.

We wanted to evaluate further if RNA deadenylation/degradation or transcription biased the evaluation of the poly(A) tail length of cyclin B1L between C 0h and W 0h. All pools of oocytes and embryos were spiked with 1 pg of exogenous GFP polyadenylated RNA [33]. Cyclin B1L was quantified in triplicate pools (n = 20) of C 0h and W 0h oocytes, and air-dried pellets were dissolved in 10 µl of ddH2O. Either 5 µM (final concentration) of Random Decamers (Ambion) or 1 µM oligo-dT12 was added and the volume adjusted to 12 µl with ddH2O. Removal of secondary structures was done by heating the mixture at 65°C for 3 min and cooling it quickly on ice for 3 min. Cold Omniscript reverse transcriptase mix (Qiagen) was added following the manufacturer recommendations. Reverse transcription (RT) was performed at 37°C for 1 h. Cyclin B1L expression was then measured using real-time PCR with the primers specific for this transcript (see Fig. 2) and as outlined above.

Experient 4: Measurement of Cyclin B1L Poly(A) Tailand Protein Expression in Cold Oocytes Matured for 4 h

In an attempt to rescue polyadenylation, cold and warm oocytes were incubated for 4 h in modified SOF medium (treatments C SOF and W SOF, respectively) [13]. The COCs were recovered and manipulated as described for treatments C 0h and W 0h except that all COCs, including treatment C 0h, were manipulated at room temperature. After maturation, oocytes (n = 20; triplicate) were denuded, washed, and frozen at –80°C until RNA extraction. Poly(A) tail length was evaluated by the polyadenylation test (RACE-PAT) described below, and protein expression of cyclin B1 was determined using Western blot analysis.

Polyadenylation Test

Measurement of poly(A) tail length of cyclin B1L (long 3'-UTR isoform) transcript on oocyte pools (n = 100; in duplicate) at the GV stage and matured for 3, 5, 8, 10, 15, 20, and 25 h was performed using a modified version of a technique called RACE-PAT [34]. Radioactive PCR products migrated on polyacrylamide gels tend to generate a smear during migration that interferes with poly(A) tail length analysis, because the smear represents the poly(A) tail length of the analyzed transcript. We optimized the technique until we were able to obtain smears with clearly defined upper and lower ends. The RACE-PAT technique is based on the annealing of an oligo-dT18 anchor to the poly(A) tail. As a consequence, the poly(A) tails that we measured are 17 adenosine residues (A) of fewer in length.

The RACE-PAT cDNA synthesis was conducted with oligo-dT anchor sequence 5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18-3' obtained from GeneRacer Kit version J (Invitrogen) and Omniscript Reverse Transcription Kit (Qiagen). The RT was done as follows: The RNA pellet, dissolved in 10 µl, was heated for 5 min at 65°C and cooled for 2 min on ice before the addition of cold reverse-transcriptase mix as recommended by the manufacturer except that 1 µl of 50 µM oligo-dT anchor was added. The RT was performed at 37°C for 1 h.

The PCR amplification was done with HotStar Taq DNA Polymerase (Qiagen) as recommended by the manufacturer. In a total reaction volume of 50 µl, cDNA equivalent to one oocyte, 1 µl of 10 µM cyclin B1 forward primer (5'-TCTTGTTGTTATGCAACACCTGGC-3'), 1 µl of 10 µM nested anchor reverse primer (5'-CGCTACGTAACGGCATGACAGTG-3'), and 0.5 µl of 10 µCi/µl of [32P]{alpha}-dATP (6000 Ci/mmol; Amersham Biosciences, Baie d'Urfé, QC, Canada) were added. The PCR amplification reaction was carried out in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA), and conditions were as follows: 1 cycle at 95°C for 15 min; 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; and a final elongation at 72°C for 7 min.

The PCR products purified with the QIAquick PCR Purification Kit (Qiagen) were migrated once with the equivalent of 15 000 cpm (volumes were adjusted) and once with 5 µl of PCR reaction per lane on a nondenaturing 3.5% Tris-borate-EDTA polyacrylamide gel at 175 V for 2 h. This double-migration was performed to verify that results were the same. Without fixation, gels were dried and exposed on a phosphor screen and revealed with Personal Molecular Imager FX (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Smears representing the poly(A) length were measured using Quantity-One software version 4.4.1 (Bio-Rad). The lane background and minimum threshold (both in terms of cpm) were taken into account. As recommended, the specificity of the amplified product was verified by digestion, and the expected band lengths were analyzed by polyacrylamide gel electrophoresis [35].

Western Blot Analysis

Pools of 100 denuded cold oocytes at time zero (C 0h) and warm oocytes at time 0 h (W 0h) were collected and frozen at –80°C until they were lysed in 2x buffer (2% [w/v] SDS, 20% [v/v] glycerol, 20 mM Tris-HCl [pH 8.8], 2 mM EDTA, and freshly added 100 mM dithiothreitol) and electrophoresed on a 12% polyacrylamide gel (experiment repeated four times). As positive controls, 15 µg of human nonstimulated A431 cell lysate (Upstate) and bovine ovary total protein lysate were migrated (see Fig. 8A). Proteins were transferred with Trans-Blot Semi-Dry apparatus (Bio-Rad Laboratories, Hercules, CA) on a 0.22-µm NitroPure membrane (Osmonics, Inc., Westborough, MA) at 15 V for 30 min with Bjerrum and Schafer-Nielson transfer buffer [36]. The membrane was stained with ponceau red to verify that protein was evenly loaded and transferred. The membrane was blocked for 1 h with 1% nonfat dried milk in PBS. After two quick rinses with PBS, mouse monoclonal clone B63 anti-goldfish cyclin B1 [37] was diluted 1:1000 in 1% milk-PBS and used for first hybridization during 3 h at room temperature. This monoclonal antibody was kindly provided by Dr. Masakane Yamashita (Hokkaido University, Sapporo, Japan). The membrane was washed twice for 10 min each time with TBSTT (500 mM NaCl, 20 mM Tris-HCl, 0.2% [v/v] Triton X-100, and 0.05% [v/v] Tween-20 [pH 7.5]) and once for 10 min with TBS (150 mM NaCl and 10 mM Tris-HCl [pH 7.5]) before second hybridization was done with peroxidase-conjugated AffiniPure goat anti-mouse immunoglobulin G (H+L; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:15 000 in 1% milk-PBS. Finally, the membrane was washed five times for 10 min each time in TBSTT before it was revealed by ECL Western Blotting Detection Reagents (Amersham Biosciences). Protein molecular weight (Mr x 10–3) was calculated with Quantity-One software version 4.4.1 (Bio-Rad).



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  		FIG. 8. Western blot showing cyclin B1 protein level in (A) human nonstimulated A431 cell lysate (Upstate) as a positive control and bovine ovary and (B) in cold GV-stage oocytes at time zero (C 0h) and warm GV oocytes at time zero (W 0h)

Statistical Analysis

Data for all experiments are presented as the mean ± SEM. Differences were considered to be statistically significant at the 95% confidence level (P < 0.05). For the experiments (triplicates) in which we quantified cyclin B1L or cyclin B1L+B1S mRNA during maturation and development and the experiments (triplicates) in which we quantified cyclin B1L mRNA in C 0h and W 0h with different types of RT (dT or decamers), data were normalized with GFP and analyzed by ANOVA with the SAS general linear model (GLM) procedure, and significant differences were obtained with the least-significant-difference (LSD) test. For the experiments in which we measured the poly(A) tail of cyclin B1L during maturation (GV stage and 3, 5, 8, 10, 15, 20, and 25 h; two replicates), the data were analyzed by ANOVA with the SAS GLM procedure, and significant differences were obtained with the Tukey Studentized Range (HSD) test. For the experiments in which we measured the poly(A) tail of cyclin B1L in treatments C 0h and W 0h (three replicates) and in oocytes from cold or warm ovaries with treatments C 0h, C SOF, W 0h, and W SOF (triplicates), the data were analyzed by ANOVA with the SAS GLM procedure, and significant differences were obtained with the protected LSD test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
3'-RACE and Sequencing

By 3'-RACE and sequencing, we were able to confirm the alternate use of the first HPS (nucleotides 28–33) (Fig. 1), by which a short 3'-UTR (cyclin B1S) of only 57 nucleotides is formed and does not contain any known cis-acting regulatory elements. When the second HPS used (nucleotides 86–91) (Fig. 1), cyclin B1 3'-UTR is 117 nucleotides long (cyclin B1L). Bovine cyclin B1L 3'-UTR sequence bears one putative CPE sequence (Fig. 1). This CPE (position 82–91) is fused to the last HPS (TTTTAATAAA). Cyclin B1L also contains a possible AU-rich element (ARE) at position 72–80. Our sequence differs from the published cyclin B1 sequence by an additional four nucleotides (TTGG) between nucleotides 1441 and 1442 (accession no. L26548).

Cyclin B1L and Cyclin B1S mRNA Levels During Maturation and Embryo Development

This experiment was conducted to elucidate if cyclin B1 mRNA is depleted over the course of maturation and/or embryo development, as expected for a maternal mRNA stockpiled in the oocyte. Cyclin B1L (long 3'-UTR) and cyclin B1S (short 3'-UTR) were amplified with primers that generate an amplicon shared by both isoforms (Fig. 2). Our results showed that cyclin B1L and cyclin B1S mRNA levels diminish between GV-stage oocytes and 4-cell embryos and between 4-cell and 8-cell embryos (Fig. 3A). When only cyclin B1L was amplified (Fig. 2), mRNA level remained stable between GV-stage oocytes, MII-stage oocytes, and 2-cell and 4-cell embryos but was decreased in 8-cell embryos and blastocysts (Fig. 3B).



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  		FIG. 3. Quantification of both cyclin B1 mRNA isoforms (A), cyclin B1L (long 3'-UTR) and cyclin B1S (short 3'-UTR), and only the longest isoform cyclin B1L (long 3'-UTR; B) in bovine oocytes and early embryos using real-time RT-PCR. Maturation and development stages studied: GV, Germinal vesicle oocytes; MII, metaphase II oocytes; 2-cell, 2-cell embryos; 4-cell, 4-cell embryos; 8-cell, 8-cell embryos; Blast, blastocysts. Each developmental stage was done in triplicate. and mRNA quantity was corrected with an exogenous GFP value obtained for each pool. Shown are relative mRNA abundance (mean ± SEM). Different letters indicate a significant difference (P < 0.05)

Polyadenylation Status of Cyclin B1L mRNA During Maturation

Because of the presence of a CPE sequence in cyclin B1L 3'-UTR, we measured poly(A) tail length of this transcript during maturation (GV stage and 3, 5, 8, 10, 15, 20, and 25 h of maturation) by RACE-PAT (Fig. 4). Unexpectedly, cyclin B1L already was polyadenylated extensively in GV-stage oocytes (150 A). The poly(A) tail was elongated between the GV stage and 10 h of maturation by approximately 50 A (Fig. 4). By overexposure of the gels, we were able to distinguish cyclin B1S at the expected height, and although it seemed to be polyadenylated, its low detection level was not suitable for quantitative analysis (results not shown).



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  		FIG. 4. Bovine oocyte cyclin B1L (long 3'-UTR isoform) poly(A) tail length during in vitro maturation. Treatments studied: GV stage and 3, 5, 8, 10, 15, 20, and 25 h of in vitro maturation. Top) Histogram of the poly(A) tail length (mean ± SEM) measured during maturation in duplicate. Different letters indicate a significant difference (P < 0.05). Middle) Representative replicate radioactive gel used to measure the poly(A) tail length shown (Top). Bottom) Time line representing bovine oocyte maturation with corresponding occurrence of GVBD, MI, and MII stages. (Modified from [13])

Polyadenylation Status of Cyclin B1L after Transportof Ovaries in Warm or Cold Conditions

This experiment was designed to assess the poly(A) tail length of cyclin B1L without the effect of ovary transport by using cold conditions to stop the polyadenylation process. Figure 5A shows results (triplicates) of bovine oocyte cyclin B1L poly(A) tail length on cold GV-stage oocytes at time zero (C 0h) and warm GV oocytes at time zero (W 0h). A significant rise (~110 A) in poly(A) tail length was found between C 0h (56 A) and W 0h (168 A) oocytes. A representative replicate is shown in Figure 5B.



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  		FIG. 5. Bovine oocyte cyclin B1L (long 3'-UTR isoform) poly(A) tail length in GV-stage oocytes transported to the laboratory on ice (C 0h) or transported in warm saline (W 0h). A) Poly(A) tail length is shown graphically. B) A representative replicate of the poly(A) tail length shown as [32P]PCR product, using a modified RACE-PAT technique, migrated on 3.5% polyacrylamide gel and revealed by phosphor imaging. Each treatment was performed in triplicate. Values are shown as the mean ± SEM. Different letters indicate a significant difference (P < 0.05)

Cyclin B1L mRNA Quantification after Transportof Ovaries in Warm or Cold Conditions

We wanted to evaluate further if RNA deadenylation/ degradation or transcription biased our evaluation of the poly(A) tail length of cyclin B1L between C 0h and W 0h. Real-time PCR was used to quantify mRNA levels of cyclin B1L mRNA between C 0h and W 0h oocytes (n = 20, three replicates) with either oligo-dT or decamer used for RT. The results showed no significant differences between mRNA levels in C 0h and W 0h treatments when either oligo-dT or decamers was used for RT (Fig. 6).



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  		FIG. 6. Quantification of cyclin B1L mRNA isoform in bovine oocytes using real-time RT-PCR. For each treatment, RT was done either with dT or decamer oligos. Each dT and decamer treatment was done in triplicates and mRNA quantity was corrected with the GFP value obtained for each pool. The relative mRNA abundance (mean ± SEM) is shown. No significant differences were found (P > 0.05)

Rescue of Cyclin B1L mRNA Polyadenylationafter Transport of Ovaries in Cold Conditions

To investigate if cytoplasmic polyadenylation could be rescued, cold oocytes were put in maturation. Poly(A) tail length was measured on oocytes from cold or warm ovaries not matured (C 0h and W 0h, respectively) or matured during 4 h in SOF medium (C SOF and W SOF, respectively) (Fig. 7). Our results show that when cold oocytes are put into maturation, cytoplasmic polyadenylation of cyclin B1L did not occur during the first 4 h of IVM. Analysis of oocytes from warm ovaries as a control showed the expected long poly(A) tail. However, we did not detect poly(A) elongation between W 0h and W SOF. As a consequence, we cannot conclude without any doubt that maturation conditions would have allowed polyadenylation to occur.



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  		FIG. 7. Bovine oocyte cyclin B1L (long 3'-UTR isoform) poly(A) tail length. Treatments studied: oocytes from cold or warm ovaries not matured (0h) or matured during 4 h in SOF (SOF; triplicate). Values are shown as the mean ± SEM. Different letters indicate a significant difference (P < 0.05)

Cyclin B1 Protein Expression after Transport of Ovariesin Cold or Warm Conditions

Because cytoplasmic polyadenylation often is correlated with translation initiation, we investigated the presence of cyclin B1 protein in cold and warm oocytes. Our results showed that when oocytes (C 0h) were collected from cold ovaries, cyclin B1 protein was never present. When oocytes (W 0h) were collected from warm ovaries and transported during 4 h, cyclin B1 protein was detectable in one of four replicates (Fig. 8B). By increasing the transportation time to 5 h, cyclin B1 protein was detectable in all two replicates of warm oocytes and was still undetectable in cold oocytes. Figure 8A shows the Western blot detection of cyclin B1 in human commercial positive-control cells and in bovine ovary. Our results showed that the antibody used against cyclin B1 detects a single band in both species at the same molecular weight.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fine-tuned posttranscriptional regulation of genes involved in the cell cycle of oocytes and embryos is of major importance. The complexity of this regulation involves many different mechanisms. We have found that bovine oocytes generate two different transcripts that both code for cyclin B1 but that bear different 3'-UTR lengths. These different isoforms are generated by the alternate use of tandem HPS (AATAAA) for 3'-end formation. This phenomenon was observed in Caenorhabditis elegans and in the mouse, where the transcript harboring the shortest 3'-UTR lacks CPE sequences and mainly is present in sperm, whereas the transcript with the longest 3'-UTR that possesses CPE sequences and other regulatory elements mainly is present in oocytes [38, 39]. This situation also could be the case in the bovine. Cyclin B1L (long) 3'-UTR is 117 nucleotides in length and possesses two HPS (AATAAA), the second of which is used for 3'-end formation (for review, see [40, 41]), and a putative CPE (Fig. 1). The CPE (TTTTAAT) overlaps the last HPS (TTTTAATAAA). A CPE overlapping an HPS also is present in X. laevis cyclin B1 (accession no. J03166) and cyclin A1 (accession no. X53745) 3'-UTR. This overlapping CPE has been called a Mos response element (MRE) [30]. In Xenopus oocytes, Mos mRNA polyadenylation and translation is necessary for cytoplasmic polyadenylation of MRE-containing mRNA, like cyclin B1, as demonstrated by antisense RNA directed against Mos [30]. Cyclin A1 mRNA is polyadenylated during maturation [21], and injection of its 3'-UTR containing a single CPE overlapping the hexanucleotide TTTTTAATAAA can be polyadenylated following maturation [30]. Additionally, it was found in an other study [42] that Xenopus cyclin B1 3'-UTR, with only its second overlapping CPE (TTTTAATAAA), also can be polyadenylated during maturation. Consequently, the bovine cyclin B1L single CPE overlapping the HPS could be sufficient to drive cytoplasmic polyadenylation.

Steady-state levels of cyclin B1L and cyclin B1 (S+L isoforms) mRNA during oocyte maturation (GV and MII stages) and embryo (2-, 4-, and 8-cell embryos and blastocysts) development was assessed by real-time PCR. Our results showed that cyclin B1L (long 3'-UTR with CPE) mRNA level remains stable from GV-stage oocytes up to 4-cell embryos and that mRNA level diminishes between 4-cell and 8-cell embryos. When both isoforms were quantified, a drop in mRNA level was seen between GV-stage oocytes and 4-cell embryos. Further studies will be needed to elucidate whether this drop results from cyclin B1S disappearance. Moreover, in the present experiment, we could not discriminate between mRNA deadenylation and degradation, because oligo-dT were used for cDNA synthesis. Whether cyclin B1S is deadenylated during maturation by a default mechanism described for maternal mRNA lacking CPE sequences is unclear (for review, see [17, 43]). Cyclin B1L contains a possible ARE at position 72–80 that is absent from cyclin B1S, and this could be implicated in polyadenylation status, RNA stability, and/or translation [44– 47]. Consequently, the stabilization of cyclin B1L mRNA could be the consequence of the presence of cis-acting elements (ARE element and CPE) in its 3'-UTR. A gene as important as cyclin B1 needs to possess many different ways to regulate and ensure its presence at the right time and quantity during oocyte maturation and embryonic development without de novo transcription. Total cyclin B1 mRNA level drops to very low levels in the 8-cell embryo and remains low in blastocysts. In porcine embryos, degradation of maternal cyclin B1 mRNA occurs over the 4-cell stage (MZT), with no transcriptional contribution from the embryo [48]. These findings support our own, in that stored maternal cyclin B1 transcript would be degraded around MZT without a detectable burst in mRNA transcription by the embryo around the MZT. If the duration of the bovine embryo fourth cell cycle is under zygotic transcriptional control [49], the steady-state level observed in 8-cell embryos could come, in part, from embryonic genome transcription. Further experiments using a transcription inhibitor could answer this question.

In bovine oocytes recovered from follicles less than 2, 3–5, and greater than 6 mm in diameter, mRNA levels of cyclin B1 and Cdk1 (p34cdc2) do not vary [11]. Furthermore, we showed that the levels of cyclin B1L mRNA in recovered bovine oocytes from 3 to 5 mm are stable during maturation. However, an earlier study conducted in our laboratory showed that cyclin B1 protein accumulates during maturation (detectable 3 h following the start of IVM) [10]. With the fact that cyclin B1L bears a CPE-like sequence and other regulatory sequences, these findings reinforced our assumption that cyclin B1L is regulated posttranscriptionally by cytoplasmic polyadenylation that would initiate its translation during maturation. Our results show that when GV-stage oocytes are collected from ovaries that were transported in warm saline for a period of 4 h, cyclin B1L transcript already is polyadenylated extensively (~150 A). This poly(A) tail is longer than expected when compared to Xenopus (~30 A) [21] and mouse (<100 A) [31] oocyte cyclin B1 mRNA poly(A) tail at the GV stage. Furthermore, comparison of the poly(A) tail length over the course of IVM (GV stage and 3, 5, 8, 10, 15, 20, and 25 h of maturation) showed that cyclin B1L transcript poly(A) tail does not undergo subsequent major changes. We observed a small rise of approximately 50 A between the GV stage and 10 h of maturation and a total poly(A) elongation of approximately 57 A between the GV- and MII-stage oocytes. Our results are supported by those of another study [50], which found that bovine oocyte cyclin B1 mRNA is not deadenylated between GV- and MII-stage oocytes. A gradual increase also was seen in endogenous mouse cyclin B1 during the course of maturation [31]. Although only the GV, MI, and MII stages were investigated in the mouse oocyte, these results are in accordance to those that we have observed in the bovine species.

From our results, we could have concluded that bovine oocyte cyclin B1 behaves differently compared to that of other species in which the cyclin B1 mRNA poly(A) tail is short in GV-immature oocytes and elongated after the start of maturation [21, 31]. However, we thought that without the effect of transportation, cyclin B1 could possess a short poly(A) tail. Furthermore, an earlier study conducted in our laboratory showed that oocytes collected from ovaries that were incubated in warm saline for 4 h (compared to 2 h) before oocyte aspiration showed increased developmental competence [51]. Thus, we investigated the status of cyclin B1L mRNA polyadenylation in oocytes aspirated from slaughterhouse cow ovaries incubated in warm saline for 4 h or transported on ice (COCs and oocytes manipulated as much as possible on ice). We excepted that the cold conditions would slow down, or even stop, cytoplasmic polyadenylation [52], which it did. Our results showed that bovine cyclin B1L mRNA in follicle (diameter, 3–5 mm)-enclosed oocytes is stored with a short poly(A) tail of approximately 56 A. Moreover, cyclin B1L is polyadenylated extensively in oocytes collected from postmortem ovaries incubated and transported in warm saline for 4 h before aspiration. Developmental competence acquired before IVM could be the result of initiation of the cytoplasmic polyadenylation, by an unknown trigger, of important stored maternal mRNA, of which cyclin B1L would be included [51; present study]. Although completely hypothetical, one trigger could be the decreased oxygen supply to the oocyte (hypoxia). Following hypoxia, Hu protein R (HuR) rapidly binds AREs in the 3'-UTR vascular endothelial growth factor mRNA (rat skeletal muscle) [53]. Interestingly, Xenopus homologue of HuR (elrA) could be implicated in cytoplasmic polyadenylation (for review, see [17, 54]). Maybe postmortem incubation of the ovaries induces hypoxia in the oocyte, enhancing HuR binding [53] to the cyclin B1L 3'-UTR ARE [45], triggering cytoplasmic polyadenylation by interacting with CPEB [55] or other proteins [17, 28, 29, 44]. The lengthening of the poly(A) tail seen between cold and warm oocytes could have been the result of RNA nuclear polyadenylation of newly synthesized transcripts [56]. However, by real-time PCR, we have shown that no difference exists in mRNA levels of cyclin B1L between cold and warm oocytes. Finally, cyclin B1L cytoplasmic polyadenylation could be correlated with initiation of translation, because cyclin B1 protein is absent from cold oocytes but present in warm oocytes. It is unclear why the presence of cyclin B1S does not lead to cyclin B1 protein presence in GV-stage bovine oocytes. Nevertheless, when cyclin B1S and cyclin B1L 3'-UTR were analyzed, a common TGTA sequence (position 53–56), known in Xenopus cyclin B1 3'-UTR to be bound by XPum (Xenopus homologue of Drosophila pumilio) [28], was found. In Xenopus, XPum physically interacts with CPEB and is a specific cyclin B1 translation regulator (repressor and initiator) during progesterone-induced maturation [28, 29]. It remains to be elucidated whether cyclin B1S mRNA is translationally repressed in GV-stage oocytes by the binding of a trans-acting factor like XPum and never unmasked because of the absence of a CPE. We conclude that cyclin B1L transcript is stored with a short poly(A) tail in a translationally inactive state and that incubation of the postmortem ovary for 4 h triggers the first burst of cytoplasmic polyadenylation, which could be responsible for translation initiation and the appearance of cyclin B1 in bovine oocytes before IVM. Further experiments will be needed to elucidate whether posttranscriptional regulation of cyclin B1L (poly(A) tail-length status) could be a potential indicator of oocyte competency.


   ACKNOWLEDGMENTS
 
We thank Dr. Masakane Yamashita (Hokkaido University, Sapporo, Japan) for his gift of mouse monoclonal anti-cyclin B1 antibody, Dr. Suzan Novak for statistical analysis, and Drs. Claude Robert and Suzan Novak for reviewing this manuscript.


   FOOTNOTES
 
1 Funded by the Natural Sciences and Engineering Research Council of Canada. Back

2 Correspondence: Marc-André Sirard, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Sainte-Foy, QC G1K 7P4, Canada. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca Back

Received: 28 July 2004.

First decision: 29 August 2004.

Accepted: 29 November 2004.


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