Piwi And PiRNA Expression And Their Relationships With Fertility Of Ploidy Cyprinids

By using red crucian carp(Carassius auratus red var.,2n=100) and common carp hybridization (Cyprinus carpio L.,2n=100), allotetraploids (F3) were formed. Base on the diploid gametes of tetraploids, the triploids were produced by intercross tetraploids with diploids. The triploids showed fast growth rates, high disease resistance which benefit for aquaculture. The tetraploids and triploids provide a good platform for study of different ploidy fishes. The present study analyzed the comparative expression of Piwi and piRNA to discussed difference and homology among triploids, red crucian carp and tetraploids. Furthermore, gonad development of teleost was mainly regulated by HPG axis. Triploids showed irregular expression of HPG axis. Thus, it is essential to discuss the regulation of HPG on Piwi-piRNA pathway. The present research provides clues for molecular endocrinology evidence for triploids and tetraploids. And the study offered reference for the roles of Piwi-piRNA during gonad development and gametogenesis in teleosts.We first compared Piwi and piRNA differential expression among red crucian carp, triploids and tetraploids. And we investigated the HPG axis regulation on Piwi and piRNA. The results include:1. Piwi gene full length cDNA cloning in red crucian carp, triploids and tetraploids, including Piwil-1and Piwil-2. The Phylogenetic tree composed by two main branches. One branch was composed by Hiwi, Xiwi, Ziwi and ect. Another was composed by Hili, Xili, Zili, and ect. All the deduced protein sequences contained PAZ and PIWI domain. In teleost, only two Piwi genes were found rather than three or four in amphibians or mammalians.2. Comparative study of differential expression in tissues of different ploidy fishes. All the Piwi genes were only detected in testes and ovaries, while no signal were found in other tissues. In ovulation period, triploids showed the lowest expression among the different ploidy fishes. In ovaries, triploids showed the highest expression among the different ploidy fishes. The Immunohistochemistry indicated that Piwil-1and Piwil-2proteins were expressed in early oocytes, follicular cells, granule cells and corona radiate of mature oocytes. In ovulation periods, the ovaries of triploids were composed by early oocytes which showed higher expression than other two fishes.3. The regulation of HPG on Piwi-piRNA pathway. Both in vivo and in vitro treatment showed that hCG down-regulated Piwi gene expression. The result suggested that LH suppresses Piwi. Similarly, E2down-regulated Piwi gene expression both in vivo and in vitro treatment. Meanwhile, triploids had the lowest LHR expression than red crucian carp and tetraploids in ovaries during ovulation. Thus, in ovulation periods, irregular HPG axis conduce low E2concentration. In total, low concentration of E2could not suppress Piwi, which lead to high expression of Piwi genes.4. By small RNA cloning in ovaries,61piRNAs,72piRNAs and66piRNAs were obtained in red crucian carp, triploids and tetraploids, respectively. Seven piRNAs were obtained in all the three fishes. We then blast the piRNAs with EST, GSS, Nucletide and liver transcriptomes of red crucian carp and common carp. The result indicated that12(6.03%) piRNAs were matched the database. The sequences included ikbkg (immune function), zp2.5(oogenesis), ttll10(microtubule protein tyrosine ligase), ttnb (integral part of the muscle fibers) and zbtb48(transcription factor) which suggest various function of the sequences.5. Differential expression of piRNA in different ploidy fishes and E2regulation on the piRNAs. In the studied7piRNAs,5piRNA showed higher expression in triploids than red crucian carp and tetraploids. The results indicated correlation between piRNA and Piwi genes. Moreover, piRNA expression was down-regulated by E2which is the terminal factor of HPG axis.In summary, in ovulation periods, factors of HPG are high expressed which depress the expression of piRNA. Low expression of piRNA releases the related sequences, and induces ovulation in teleosts. In sterile triploids, the low expression of LHR conduce high expression of Piwi-piRNA, and finally result in sterile of triploids. The present research offered new information for piRNA, and discussed the affection of HPG axis. The results provide data for molecular endocrinology of teleosts.