Tale of the Human Oocyte Poly(A) Tail

OBJECTIVE: Resumption of meiosis in oocytes of all species studied to date is marked by a virtual cessation of transcription and approximately 50% of the polyadenylated RNA is degraded or deadenylated to a stable form and readenylated when required later in development. Oocytes that fail to accumulate and regulate the RNA transcribed during development and maturation will result in an incorrect temporal utilization of maternal message and would be expected to exhibit abnormal preimplantation development. The objective of the present study was to evaluate Poly(A) tail length of selected genes in oocytes matured in vitro (IVM) compared to oocytes matured in vivo (MII). DESIGN: An institutional ethics committee approved human research study conducted in a university setting with collaborative partners from private ART clinics. MATERIALS AND METHODS: Oocytes were donated by women undertaking ART for infertility. Germinal vesicle stage (GV) and MII oocytes were recovered from gonadotrophin stimulated cycles and prepared within 6 hrs of collection for RNA analysis. In addition a proportion of the GV oocytes were matured in vitro to IVM oocytes and prepared for RNA analysis. The four cell cycle genes, CENPE, AURKA, BUB1 and MAD2L1 were selected for detailed molecular analysis for Poly(A) tail lengths. Poly(A) transcripts were G-tailed, reverse transcribed using a C12T6 primer and then amplified in two rounds using nested gene specific primers close to the 3′ end in combination with the C12T6 primer. PCR products were purified and cloned into a TA vector and transfected into E. coli. Inserts were individually selected and sequenced to determine the exact number of A nucleotides in the Poly(A) tail. RESULTS: For all genes examined the Poly(A) tail length was significantly longer in the IVM oocytes compared to MII oocytes (P<0.01). Additionally, with the exception of AURKA, all genes examined had a significantly longer Poly(A) tail length in IVM oocytes compared to the GV oocytes from which these were derived (P<0.001). CONCLUSIONS: For the cell cycle genes examined, IVM oocytes have a significantly longer Poly(A) tail than MII oocytes indicating dysregulation of the normal mRNA processing that occurs during maturation in vivo. Failure to deadenylate or precocious polyadenylation of genes in IVM oocytes may explain the basis of the developmental incompetence of these oocytes.