Offers a very good model for analyzing the molecular mechanisms underlying
Offers a very good model for analyzing the molecular mechanisms underlying the ovarian differentiation in teleosts. Previously, we have analyzed the gonadal development of ricefield eel larvae through histological observation, and shown that the indifferent gonad differentiated into the ovary at 7 dph [25]. However, the regulatory gene networks and mechanisms of ovarian differentiation in ricefield eels remain to be explored.The next-generation high-throughput sequencing technology has been employed to identify genes involved in gonadal development, sex determination and sex differentiation [26, 27]. In the present study, large-scale transcriptomic analysis of gonads of ricefield eel larvae around ovarian differentiation was performed with the intention of revealing the genes and gene networks involved in the early ovarian differentiation. It is suggested that up-regulation of female-related genes and low expression of male-related genes in gonads of ricefield eel larvae participate in the cascade of sex differentiation leading to the final female phenotype. The up-regulation of genes for synthesis of retinoid acid (RA) and low expression of genes for RA degradation may be a prerequisite for adequate production of RA and subsequent initiation of meiosis in the differentiated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28388412 ovaries of ricefield eel PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 larvae. Results of present study provide a solid foundation for further elucidation of the mechanisms of the ovarian differentiation in ricefield eels and other teleosts as well.MethodsExperimental fish and sample collectionThe adult ricefield eels (females:body length 35?5 cm and body weight 35?0 g;males: body length 45?0 cm and body weight 90?50 g) were purchased from Dazhong Breeding Co. Ltd. (Jianyang, Sichuan, China), and raised in outdoor concrete ponds under natural photoperiod (30?9 ) and temperature (21.5?2.5 ) feeding on live Chironomus sp. larvae. In May 2015, mature male and female fish were selected for mating and newly hatched ricefield eel larvae were obtained for the present study. The ricefield eel larvae were fed with finely tubificidae starting at 5 days post hatching (dph), and were collected at 6, 9, 12, and 20 dph for analysis, with the average body length (cm) of 2.44 ?0.15, 3.31 ?0.09, 4.07 ?0.14 and 5.17 ?0.14, respectively, and average body weight (g) of 0.0126 ?0.0017, 0.0197 ?0.0018, 0.0392 ?0.0032 and 0.0600 ?0.0047, respectively. At each time point, 3 larvae were fixed in Bouin solution for 24 h and then stored in 70 ethanol until histological examination of gonadal stages, and 6 larvae were kept in Sample Protector for RNA/DNA (RP54476 site Takara Biotechnology, Dalian, China) for the extraction of tissue RNA.Histological examination of gonadal stages of ricefield eel larvaeThe Bouin-fixed ricefield eel larvae were decalcified in Fisher’s Cal-Ex solution (Thermo Fisher Scientific, USA) overnight and then washed with running tap water for 24 h before conventional histological processing. The whole body of ricefield eel larvae was embedded in paraffin. Serial cross sections were cut at 5 m and stainedCai et al. BMC Genomics (2017) 18:Page 3 ofwith haematoxylin/eosin. Micrographs were taken under a microscope (E800, Nikon, Japan).RNA extraction from gonadal tissues of ricefield eel larvaeThe ricefield eel larvae were kept in Sample Protector for RNA/DNA (Takara) and the gonadal tissue was dissected under a dissecting microscope (SteREO Lumar, V12, Zeiss, Germany. Additional file 1: Figure S1). Gonadal tissues were pooled to.