Research paper Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination Wenteng Xu, Hailong Li, Zhongdian Dong, Zhongkai Cui, Ning Zhang, Liang Meng, Ying Zhu, Yang Liu, Yangzhen Li, Hua Guo, Jialu Ma, Zhanfei Wei, Nianwei Zhang, Yingming Yang, Songlin Chen ⁎ a Yellow Sea Fisheries Research Institute, CAFS, Key Lab for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao 266071, China b Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China abstract article info Article history: Received 10 March 2016 Received in revised form 20 July 2016 Accepted 25 July 2016 Available online 30 July 2016 E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participa- tion in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcrip- tion of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis. © 2016 Elsevier B.V. All rights reserved. Keywords: Cynoglossus semilaevis neurl3 Expression pattern RNA interference Spermatogenesis Ubiquitination 1. Introduction Half-smooth tongue sole (Cynoglossus semilaevis) is an economically important marine fish widely cultivated in northern parts of China. This fish exhibits significant sexual dimorphism, e.g. females usually grow 2–4 times larger than males. However, in aquaculture populations the female proportion is usually very low and this substantially reduces industrial productivity. There are many reasons behind the low female ratio in aquaculture, an important one of which is the frequent occur- rence of sex-reversal. The half-smooth tongue sole has a ZW/ZZ sex determining system and the females contain heterogametes (female: ZW; male: ZZ), although under some conditions sex-reversal occurs and genetic female fish become phenotypic male fish, designated as pseudomale (ZW). The pseudomale fish can develop mature testis, produce sperm and have male growth characteristics (Chen et al., 2014; Shao et al., 2014), and so is disadvantageous in aquaculture. Nonetheless if pseudomales are crossed with normal female, this should produce super females (WW), which can be crossed with normal male (ZZ) for full-female offspring. Attempts to generate super females have been unsuccessful due to the phenomenon of W sperm absence since the pseudomales only produce Z sperm (Chen et al., 2014). Characteri- zation of the molecular mechanism behind spermatogenesis in male and pseudomale fish should provide insight into this intriguing phenomenon. The availability of the half-smooth tongue sole genome has facilitated studies of gene function and led to the proposal that male- determining genes are mainly localized on the Z chromosome (Chen et al., 2014). Several Z chromosome localized genes have proved to be important players in sex determination, sex differentiation and spermatogenesis, such as doublesex and mab-3 related transcription factor 1 (dmrt1) and testis-specific protein kinase 1 (tesk1)(Chen et al., 2014; Meng et al., 2014). Neuralized E3 ubiquitin protein ligase 3(neur13) was identified on the Z chromosome and proposed as an intriguing candidate in spermatogenesis since it was highly expressed in testis at 116 days post hatch after gonad differentiation (Chen et al., 2014). Furthermore, the ubiquitination pathway is a conserved and complex regulatory network that participates in many physiological processes including spermatogenesis (Pickart, 2001; Shmueli and Oren, 2005; Dikic et al., 2009; Jiang and Chen, 2012). Several E3s have been suggested to function in spermatogenesis, including huwe1 that is involved in spermatogenesis by regulating histone ubiquitination and modification; mouse ubr2 that interacts and metabolically stabilizes Gene 592 (2016) 215–220 Abbreviations: A, adenosine; Bp, base pair(s); C, cytidine; G, guanosine; min, minute; neurl3, neuralized E3 ubiquitin protein ligase 3; PCR, polymerase chain reaction; RNAi, RNA interference; s, second; T, thymidine. ⁎ Corresponding author. E-mail address: chensl@ysfri.ac.cn (S. Chen). http://dx.doi.org/10.1016/j.gene.2016.07.062 0378-1119/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene