Comparative Genomic Analysis of Follicle-Enclosed Oocyte Maturational and Developmental Competence Acquisition in Two Lower Vertebrates, Oncorhynchus mykiss and Xenopus laevis.

Abstract

Maturational competence is defined as the oocyte ability to resume meiosis while developmental competence is defined as the oocyte ability to be fertilized and to subsequently develop into a normal embryo. In vertebrates, late oogenesis is a key period for the acquisition of both maturational and developmental competence. Throughout late oogenesis, several genes exhibit a differential expression in both oocyte and follicular cells to ensure proper oocyte maturation or to store transcripts and proteins that will act as maternal factors during early embryogenesis. However, the molecular mechanisms involved in these processes are far from being fully understood. In order to identify key conserved mechanisms in lower vertebrates, we performed a comparative genomic analysis using a teleost, the rainbow trout (Oncorhynchus mykiss) and an amphibian, the South African toad (Xenopus laevis). Comparison of competence acquisition mechanisms between fish and amphibians is really valuable as they share common physiological mechanisms and similar follicular structure but are distant from nearly 100 millions years. Our study aimed at identifying differentially expressed genes during late oogenesis in both species. Ovarian rainbow trout transcriptome was studied during late vitellogenesis, post-vitellogenesis and oocyte maturation using a 9K cDNA microarray. Using Significance Analysis of Microarray of hybridization signals with a False Discovery Rate of 5%, we identified 1200 differentially expressed genes. A subset of candidate genes exhibiting the most differential expression was kept for validation by real-time PCR in both species. For Xenopus, ovarian follicles at stage IV (late vitellogenesis), immature stage VI (post-vitellogenesis) and after in vitro maturation were used. In addition, genes exhibiting a lower differential expression and possibly involved in maturational or developmental competence acquisition according to the literature were also selected. As a first step, we focused on 27 genes and thoroughly analyzed the orthology relationship between trout and Xenopus sequences in order to identify true Xenopus orthologs of the identified trout genes. These genes are involved in various functions such as steroidogenesis, post-translational modifications, and cell cycle. For instance, cmah, the cytidine monophosphate-N-acetylneuraminic acid hydroxylase, exhibited decrease of mRNA expression at the time of oocyte maturation, in both species. In addition, we identified 4 genes, cyp19a1, cyp17a1, star and hsd11b3 that are important for the steroidogenic control of follicular differentiation in both species. cyp19a1 expression decreased dramatically during post-vitellogenesis in both species. cyp17a1 and hsd11b3 expression decreased, to a lower extent, during the same period. In contrast, a sharp and concomitant increase of star expression was observed in both species. In summary, we have successfully identified orthologous genes exhibiting conserved expression profile in the late oogenetic ovarian follicle of trout and Xenopus. These genes have pointed out molecular mechanisms possibly conserved in lower vertebrates. It should also be stressed that in addition to the already documented participation of steroidogenesis in maturational competence acquisition, our approach has also shed light on new mechanisms of maturational and developmental competence acquisition that we are currently further investigating. Supported by ANR-08-GENM-033 OSCILE. (poster)