Sex-Biased miRNAs in Gonad and Their Potential Roles for Testis Development in Yellow Catfish

et al. (2014) Sex-Biased miRNAs in Gonad and Their Potential Roles for Testis Development in Yellow Catfish. PLoS ONE 9(9): e107946. doi:10.1371/journal.pone.0107946 Sex-Biased miRNAs in Gonad and Their Potential Roles for Testis Development in Yellow Catfish Jing Jing 0 Junjie Wu 0 Wei Liu 0 Shuting Xiong 0 Wenge Ma 0 Jin Zhang 0 Weimin Wang 0 Jian-Fang Gui 0 Jie Mei 0 Liang-Hu Qu, Sun Yat-sen University, China 0 1 College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Huazhong Agricultural University , Wuhan , China , 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences , Wuhan , China Recently, YY super-male yellow catfish had been created by hormonal-induced sex reversal and sex-linked markers, which provides a promising research model for fish sex differentiation and gonad development, especially for testis development. MicroRNAs (miRNAs) have been revealed to play crucial roles in the gene regulation and gonad development in vertebrates. In this study, three small RNA libraries constructed from gonad tissues of XX female, XY male and YY super-male yellow catfish were sequenced. 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. 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 17a-ethinylestradiol (EE2), high dose of which will impair 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 miRNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and identifies a number of sex-biased miRNAs, some of that are potentially involved in testis development and spermatogenesis. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All small RNA data has been deposited into the NCBI Gene Expression Omnibus database (Database ID: GSE54610). Funding: This work was supported by grants to Jie Mei from the Fundamental Research Funds for the Central Universities (52204-12018, 2013PY068) and the National Natural Science Foundation of China (31301931); and to Jian-Fang Gui from the special Fund for Agro-scientific Research in the Public Interest from the Ministry of Agriculture of China (2009030406), the National Key Basic Research Program (2010CB126301). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. microRNAs (miRNAs), a class of small non-coding RNAs (,1826 nt), have been known to be involved in mRNA degradation and post-transcriptional repression [1]. Most mature miRNA sequences are conserved among fish, amphibians, birds and mammals[2]. miRNAs have been revealed to play important roles in many biological processes, such as tissue development, cell proliferation and differentiation [3]. In vertebrates, a subset of miRNAs, such as miR-430 and miR-196 are specifically expressed and functioning during early embryonic development [4,5]. Fish miR-430 regulates early primordial germ cell development by regulating sdf1a, cxcr7, TDRD7, nanos1 and c1q-like expression [69]. In adults of chicken and cattle, some miRNAs have been identified abundantly expressed in gonadal tissues [10,11]. Let-7 regulates ageing of the Drosophila testis stem-cell niche by targeting IGF-II messenger RNA binding protein [12]. However, the regulatory and functional roles of miRNAs in gonad development have not been clear in teleosts yet. In aquaculture, many fish species display significantly different growth rate between male and female. For example, in yellow catfish (Pelteobagrus fulvidraco), Nile tilapia (Oreochromis niloticus), African catfish (Clarias gariepinus), and channel catfish (Ictalurus punctatus), male exhibits much faster growth rate than female sibling [1316]. While female growth much faster than male in some other aquaculture fishes, such as gibel carp (Carassius auratus gibelio Bloch), rainbow trout (Oncorhynchus mykiss) and half-smooth tongue sole (Cynoglossus semilaevis) [17 19]. The productions of mono-sex groups of fish can be accomplished by sex-reversal technology and subsequently improve fish production. Recently, YY super-male yellow catfish was successfully created by crossing XY male with hormonal-induced XY female, and then identified by sex-linked SCAR markers [20 22]. However, the molecular mechanism of gonad development is unknown in yellow catfish. The miRNA expression profile in male and female gonad of yellow catfish has not been explored. Here, we performed deep sequencing using solexa technology on 3 types of gonad (XX females, XY males and YY males). Also, we investigated miRNA expression using quantitative real-time PCR (RT-qPCR). We found sexually dimorphic expression of many miRNAs, some of which have also been observed to have a sex-dependent expression pattern in other vertebrates. Our results indicate a wide conservation of miRNAs in teleost and suggest their possible roles in vertebrate testis development. Materials and Methods Fish samples Experiments were performed on one-year-old yellow catfish individuals (four females, four males and three YY super-males) with the same age and under the same culturing conditions. Their sex was confirmed by histological analysis and PCR with sexlinked primers as described previously [23]. The males and supermales are of the same size and weight. Experimental protocols used here were approved by the institution animal care and use committee of Huazhong Agricultural University. Hematoxylin and eosin (HE) staining was performed according to previously described [24]. RNA isolation, small RNA library preparation and sequencing Gonad tissues were taken from each individual of 4 XX females, 4 XY males, 3 YY super-males. To prevent potential cell contamination, the gill of healthy yellow catfish was cut to remove most of the blood, and the gonad tissues were washed with fresh PBS for three times. Total (...truncated)