Expression And Functional Study Of MiR-200 Family During Testis And Embryo Development Of Fish

MicroRNAs(miRNAs) have been revealed to play crucial regulatory roles in the sex differentiation and gonad development in vertebrates.Yellow catfishrepresents as one of the Chinese economical fish species with sexual dimorphism.However, the miRNA expression profile in male and female gonad has not been explored, and the molecular mechanism of gonad development is unknownin yellow catfish. Here, we performed deep sequencing using solexa technology on gonad of 3 types of yellow catfish(XX females, XY males and YY males). The sequencing data generated a total of 384 conserved miRNAs and 113 potential novel miRNAs, among which 23, 30 and 14 miRNAs were specifically detected in XX ovary, XY testis, and YY testis, respectively.There are some sex-biased expressed miRNAs in yellow catfish that have also been shown to have a sex-dependent expression pattern in other vertebrates. We observed relative lower expression of several miR-200 family members, including miR-141 and miR-429 in YY testis compared with XY testis. Histological analysis indicated a higher degree of testis maturity in YY super-males compared with XY males, as shown by larger spermatogenic cyst, more spermatids and fewer spermatocytes in the spermatogenic cyst. Moreover, five miR-200 family members were significantly up-regulated in testis when treated by high dose of 17α-ethinylestradiol(EE2) that impairs testis development and cell proliferation. The down-regulation of miR-141 and 429 coincides with the progression of testis development in both yellow catfish and human. At last, the expression pattern of nine arbitrarily selected miRNAs detected by quantitative RT-PCR was consistent with the Solexa sequencing results. Our study provides a comprehensive mi RNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and suggests that miR-200 family is potentially involved in testis development and spermatogenesis.In vertebrates, growth hormone/insulin-like growth factor(GH/IGF) axis signaling plays a critical role in regulating somatic growth. The elevated expressions of GH/IGF genes showed to be positively correlated with faster growth rate in mammalians and aquaculture fish species. However, the direct regulation of GH/IGF axis genes during early development is still unclear. The miR-200 family is highly conserved in vertebrates, with miR-8 being the sole homolog in Drosophila melanogaster.MiR-8/200 s activity in somatic growth was first discovered in Drosophila and characterized as a growth-promoting factor.However, the function of miR-200 in body growth has not been reported in vertebrates.Our studies of the zebrafish reveal that the conserved miR-200 family members are critical regulators of embryo size by targeting several GH/IGF axis genes, including GH, GHRa, GHRb and IGF2 a. Overexpression of miR-200 s led to cell cycle arrest in the G1 phase and induced apoptotic response during embryo development, thereby inhibiting somatic growth of zebrafish embryo. Intriguingly, GH induced expression of both p53 and miR-200 s, and miR-200 s are potential p53 transcriptional targets, thus forming a negative feedback loop. Significantly, the up-regulation of miR-200 s associated with GH activation is abolished in embryos with p53 mutation. By integrating these studies, we conclude that p53/miR-200 and GH/IGF signaling pathway form a negative regulatory loop to control embryo size, that provide critical insights into the long-standing puzzle of how body growth is determined during early development of teleost.