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

Abstract

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.

Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

“…The embryo has to divide thrice to reach maternal zygotic transition (MZT) in conditions of very low transcription (Barnes & First 1991). Therefore, the competent oocyte must store enough mRNA coding for cell cycle proteins like CCNB1 (Tremblay et al 2005) to ensure that these proteins will not be limiting the embryo progression.…”

RNA Processing During Early Embryogenesis: Managing Storage, Utilisation and Destruction

“…The presence of a specific sequence on all transcripts is insufficient to meet the requirements of gradual use throughout the embryonic program. The time dependant constraint of transcript utilization could be met if isoforms bearing different 3'UTR with different regulatory motifs are present in the maternal pools (Tremblay et al 2005;Pique et al 2008). It has been proposed that RNA accumulation during oogenesis may involve the production of a heterogenic RNA population.…”

Opportunities and challenges in applying genomics to the study of oogenesis and folliculogenesis in farm animals

“…Distinct RNAs bear polyA tails of distinct lengths and many maternal RNAs are regulated during maturation and early embryonic development through shortening/ elongation of the polyA tail (Tremblay et al 2005). One of the issues is that the priming reaction for gene expression studies will generate different gene expression profiles based on the length of the polyA tails.…”

“…For example, the use of poly-dT beads or membranes to capture and purify poly-A RNA will often lead to a partial evaluation of the actual transcript content: the longer the poly-A tail, the more likely that the transcript will be retained, creating a bias against short poly-A tail transcripts. Also, preferential amplification of transcripts with a long versus short polyA tail is expected when RT is primed with oligo(dT; as cyclin B, Tremblay et al 2005), whether for cDNA or RNA amplification. On the other hand, if only the long poly-A tails are associated with active translation, then such profiling would favour (if not be strictly limited to) active transcripts in the considered biological situation and possibly a link to function when isolating specific subpopulations of mRNA.…”