Biomedical subjects

Christian Vigneault

Publications and source records attributed to Christian Vigneault.

8 recordsLinked to original sources

PubMed2007

3'5'-cyclic adenosine monophosphate-dependent up-regulation of phosphodiesterase type 3A in porcine cumulus cells.

The means by which cumulus cells react to gonadotropin stimulation and regulate the subsequent production and degradation of cAMP are largely unknown. In this article, we report that cyclic nucleotide phosphodiesterase (PDE) type 3A (Pde3a) is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway during in vitro maturation (IVM). cAMP-PDE activity was increased in the cumulus-oocyte complex (COC) after 10 h of IVM, and 78% of this increase was sensitive to a Pde3-specific inhibitor, cilostamide. Although no variation was observed in the oocyte, cilostamide-sensitive cAMP-PDE activity increased in the cumulus cells after IVM. This was supported by Western blotting, which showed that the intensity of a 135-kDa anti-Pde3a immunoreactive band was increased in COC after IVM. The Pde3a mRNA level was up-regulated 28-fold in the COC after 4 h of IVM and remained high up to 12 h. The mRNA up-regulation and increased activity were inhibited by an RNA synthesis inhibitor, alpha-amanitin. The cilostamide-sensitive increase in PDE activity was inhibited by a protein synthesis inhibitor, cycloheximide. Pregnant mare serum gonadotropin (PMSG) caused dose-dependent activation of Pde3. The PMSG-dependent increase in Pde3 activity and Pde3a mRNA were mimicked by the adenylyl cyclase activator forskolin or prostaglandin E2. PMSG-dependent Pde3 activation was inhibited by the protein kinase A-specific inhibitor H89. Collectively, our results show for the first time that degradation of the intracellular cyclic nucleotide by Pde3a is transcriptionally up-regulated via a cAMP-dependent pathway in cumulus cells, suggesting that it has a functional role during the ovulatory gonadotropin surge.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2007

Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early development.

The requirement for sample standardization is basic to any relative RNA abundance assessment. In 2002, we published on the RNA abundance profiling of several housekeeping targets during early bovine embryonic development. It was then concluded that histone H2a was the most stable transcript across the studied developmental period. Since that time, several teams have applied this information, yet neglected to use the published set of primers. Here, we show that these other primer sets do not target the same histone H2a variant. Within the present report, the RNA abundance profiles of their respective targets, for example, histone H2a.1, H2a.z, and H2a.o were measured in developmental series spanning the immature oocyte to the blastocyst stage embryo. In order to more clearly define the conditions that impact the RNA abundance level measurement of these candidates, the state of polyadenylation and the origin of the transcript either from de novo transcription or from maternal stocks were taken into consideration. The histone H2a.z transcript is principally regulated by de novo transcription following embryonic genome activation, whereas the levels of H2a.1 and H2a.o variants are largely of maternal origin, conferring a more stable profile throughout the studied developmental window. The histone H2a.z was found solely in a polyadenylated state whereas the H2a.1 and H2a.o were found to be more abundant in a nonadenylated form. The presence of serum in the in vitro embryo production system also had some impact on the histone H2a.1 RNA level at the blastocyst stage.

Animals↗

PubMed2006

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos.

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.

Animals↗

PubMed2006

Identification and characterization of a novel bovine oocyte-specific secreted protein gene.

The oocyte transcriptome and proteome largely remain a mystery and the important roles of these genes, especially in the bovine, are poorly understood. To better understand specific developmental roles of the bovine oocyte, we sought to characterize a novel oocyte gene. We have sequenced the full-length mRNAs of bovine oocyte-secreted protein 1 (OOSP1) and discovered two splice variants, namely OOSP1_v1 (variant 1) and OOSP1_v2 (variant 2). The OOSP1 gene is located on chromosome 15 and is composed of 5 exons, which span over nearly 7.9 kb. Bovine OOSP1_v1 encodes a predicted protein of 163 aa in length that shares amino acid identity with mouse OOSP1, it also bears a putative signal peptide and is probably glycosylated. Bovine OOSP1_v2 protein bears the same signal peptide as OOSP1_v1 but due to the skipping of exon 2 that introduces a frameshift, it is only 35 aa in length. Expression analysis of both OOSP1 transcripts showed that this gene is exclusively expressed in oocytes, contrasting with the mouse where Oosp1 is also expressed in the spleen and liver. OOSP1_v1 and OOSP1_v2 mRNA expression profiling using real-time PCR revealed that their mRNA levels were high in germinal vesicle (GV) stage oocytes and gradually decreased until the blastocyst stage, at which time they were undetectable. For the first time, we have identified the bovine OOSP1 gene as being one of the few known oocyte-specific markers.

Alternative Splicing↗

PubMed2006

Characterization of linker histone H1FOO during bovine in vitro embryo development.

Linker histones H1 are involved in various mechanisms, such as chromatin organization and gene transcription. In different organisms, a unique subtype can be found in the oocyte, however its function remains unclear. To assess the potential involvement of this oocyte linker histone (H1FOO) in chromatin modulation, we have cloned and sequenced the bovine H1FOO cDNA and followed its mRNA profile by quantitative RT-PCR in the oocyte and throughout bovine early embryo development. The highest level of mRNA was found in the germinal vesicle (GV) oocyte and diminished constantly throughout embryo development. In the 16-cell embryo and blastocyst, respectively, the mRNA levels were 200 and 2,000 times lower than in the GV oocyte. A specific antibody raised against bovine H1FOO was used to establish protein distribution in the oocyte and preimplantation embryo by immunocytochemistry. In the GV and metaphase II (MII) oocyte, as well as in the 1-, 2- and 4-cell embryo, H1FOO was localized in the cytoplasm and nucleus. The protein was uniformly spread within the cytoplasm, while it was concentrated onto the chromatin in the nucleus. In the 8- to 16-cell embryo, H1FOO's presence diminished in the cytoplasm, although it was still strongly expressed in nucleus. In the morula and blastocyst stages, the protein was totally lacking. By its position on chromatin, H1FOO could not only be involved in chromatin conformation but could also participate in activation or repression of genes during oogenesis and embryo development before embryonic genome activation.

Amino Acid Sequence↗

PubMed2005

RNA interference as a tool to study gene function in bovine oocytes.

RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.

Adenine↗

PubMed2004

Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte.

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.

Animals↗

PubMed2004

Transcription factor expression patterns in bovine in vitro-derived embryos prior to maternal-zygotic transition.

Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

Animals↗