Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes.

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation.

While it is generally well accepted that the ovarian follicular sites of estradiol-17ß (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.

Comparative transcriptomic analysis of follicle-enclosed oocyte maturational and developmental competence acquisition in two non-mammalian vertebrates.

BACKGROUND: In vertebrates, late oogenesis is a key period during which the oocyte acquires its ability to resume meiosis (i.e. maturational competence) and to develop, once fertilized, into a normal embryo (i.e. developmental competence). However, the molecular mechanisms involved in these key biological processes are far from being fully understood. In order to identify key mechanisms conserved among teleosts and amphibians, we performed a comparative analysis using ovarian tissue sampled at successive steps of the maturational competence acquisition process in the rainbow trout (Oncorhynchus mykiss) and in the clawed toad (Xenopus laevis). Our study aimed at identifying common differentially expressed genes during late oogenesis in both species. Using an existing transcriptomic analysis that had previously been carried out in rainbow trout, candidate genes were selected for subsequent quantitative PCR-based comparative analysis. RESULTS: Among the 1200 differentially expressed clones in rainbow trout, twenty-six candidate genes were selected for further analysis by real-time PCR in both species during late oogenesis. Among these genes, eight had similar expression profiles in trout and Xenopus. Six genes were down-regulated during oocyte maturation (cyp19a1, cyp17a1, tescalcin, tfr1, cmah, hsd11b3) while two genes exhibited an opposite pattern (apoc1, star). In order to document possibly conserved molecular mechanisms, four genes (star, cyp19a1, cyp17a1 and hsd11b3) were further studied due to their known or suspected role in steroidogenesis after characterization of the orthology relationships between rainbow trout and Xenopus genes. Apoc1 was also selected for further analysis because of its reported function in cholesterol transport, which may modulate steroidogenesis by regulating cholesterol bioavailability in the steroidogenic cells. CONCLUSIONS: We have successfully identified orthologous genes exhibiting conserved expression profiles in the ovarian follicle during late oogenesis in both trout and Xenopus. While some identified genes were previously uncharacterized during Xenopus late oogenesis, the nature of these genes has pointed out molecular mechanisms possibly conserved in amphibians and teleosts. It should also be stressed that in addition to the already suspected importance of steroidogenesis in maturational competence acquisition, our approach has shed light on other regulatory pathways which may be involved in maturational and developmental competence acquisitions that will require further studies.