Identification And Expression Analysis Of Sex-related MiRNAs In Swamp Eel And Zebrafish

Teleost fish as the most ancient vertebrate represents approximately half of the vertebrate species. Study of fish reproductive strategies may provide theoretical guidance for elucidating speciation and evolution of vertebrates, and practical implications to develop techniques for artificial control of fish sex. MicroRNAs (miRNAs) are an abundant family of small regulatory RNAs, which are endogenous non-coding molecules with about 22 nucleotides in length. The mature miRNAs exert down-regulation role at post-transcriptional level by degradation mRNA or by translation inhibition. The miRNA pathways are involved in regulations of reproductive process, expanding from germ cell biogenesis, gametogenesis to fertilization and embryo development.The swamp eel(Monopterus albus) is a good model for comparative genomic studies and sexual determination/differentiation, for the reason of its primitive evolutionary status and natural occurring sex reversal from female into male via intersex during its life cycle. In this study, the miRNome during the gonadal differentiation and the differential expressions of gonadal miRNAs were analyzed by high-throughput sequencing and miRNA array in swamp eel and zebrafish (Danio rerio). The main results are as follows:1. Firstly, we obtained 1119 mature miRNAs in gonads of the swamp eel by Illumina Solexa sequencing.195 miRNAs were conserved and 924 miRNAs were novel. The 195 miRNAs were classified as 133 conserved miRNA genes which located on 234 pre-miRNAs. Among them,65 miRNA genes located on more than one pre-miRNAs. The conserved miRNAs were clustered into 84 miRNA families, most of which were evolutionarily conserved in vertebtates.924 novel miRNAs were classified as 791 miRNA genes, which located on 1372 pre-miRNAs.2.180 from 195 conserved miRNAs were co-expressed among ovary,ovotestis and testis in swamp eel, while 42 miRNAs were intersex-biased (23.33%),12 miRNAs were male-biased (6.67%) and 13 miRNAs were female-biased (7.22%). Analysis of differential expression showed that the miRNAs become active during the gonadal-reversal, especially in the prosess of testis restructuring from the ovotestis.3. miRNA array analysis showed 123 differentially expressed miRNAs in mature ovary and testis of zebrafish. By contrast,77 miRNAs were co-expressed in gonads of both fishes.10 miRNAs were expressed abundantly, and 57 miRNAs of 77 co-expressed miRNAs exhibited coincident expression trends between the ovary and testis, while 20 miRNAs displayed opposite tendencies. In situ hybridization of miR-125b, miR-205 and miR-130c showed positive signals in developing ovary and different types of sperm cells during spermatogenesis with a certain difference in expression.4. Quantitative realtime PCR indicated that miR-17-92 cluster are ovary biased expressed in both two fishes. Expressions of six members in miR-17-92 cluster were upregulated during the embryogeny of zebrafish. Downstream target genes of miR-17-92 cluster were further analyzed through bioinformatic tools, and two direct target genes involved in the process of gonad differentiation were identified by luciferase reporter assay, in which Wnt4a was down-regulated by miR-17a/20a and Dmrtl was down-regulated by miR-19a/19b.