Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

BackgroundMicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia.ResultsWe identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish.ConclusionsThe sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2636-z) contains supplementary material, which is available to authorized users.

Cellular RNA dynamics are closely associated with a vast range of physiological processes that are mostly long-lasting. To uncover the association between RNA dynamics and these processes, fluorescent RNA probes with high specificity, photostability, and biocompatibility are compulsory. Herein, a series of fluorescent carbon dots (CDs) have been prepared by one-pot hydrothermal treatment of o-, m-, or p-phenylenediamines with triethylenetetramine. Only CDs derived from the meta precursor ( m-CDs) with excellent photostability and biocompatibility can specifically bind to cellular RNA, allowing successfully long-term (up to 3 days) monitoring of RNA dynamics during cell apoptosis, mitosis, and proliferation. This RNA affinity can be attributed to the isoquinoline moieties and amines on the surface of m-CDs, which can bind to RNA through π-π stacking and electrostatic bonding, respectively. The cellular internalization of m-CDs is time-, temperature-, ATP-, caveolar, and microtubule-dependent. Additionally, investigations on the in vivo behavior of m-CD suggest that they can be efficiently and rapidly excreted from the zebrafish larvae body after 48 h. Our results provide a powerful tool for clarifying complex relationships between RNA dynamics and basic biological processes, disease development, or drug interactions.

The dmrt6 gene has been isolated from tetrapods and recently from a coelacanth, Latimeria chalumnae. Its evolutionary history and exact function remain unclear. In the present study, dmrt6 was isolated from Perciformes (five cichlids and stickleback), Siluriformes (southern catfish), and Lepisosteiformes (spotted gar). Syntenic and phylogenetic analyses indicated that dmrt6 experienced gene transposition after the divergence of teleosts from other bony fish as gene loci surrounding dmrt6 were conserved among teleosts (but was completely different from gene loci surrounding dmrt6 in tetrapods and spotted gar), while these gene loci were conserved among nonteleost species. Real-time PCR and in situ hybridization revealed that dmrt6 was highly expressed in the XY gonads from 90 days after hatching (dah) onward and was observed exclusively in spermatocytes of the testes in tilapia. Dmrt6 knockout by CRISPR/Cas9 resulted in fewer spermatocytes, down-regulated Cyp11b2 in testes, and consequently produced a lower level of serum 11-ketotestosterone (11-KT) in Dmrt6-deficient XY fish compared with the XY control at 120 dah. From 150 to 180 dah, spermatogenesis gradually recovered, and cyp11b2 expression and serum 11-KT level were restored to the same levels as those of the XY control fish. In addition, a Dmrt6 mutation was observed in genomic DNA of sperm of G0 mutant fish and F1 fish. Taken together, our data suggest that dmrt6 also exists in bony fish. Its absence in most fish genomes was probably due to incomplete sequencing and/or secondary loss. The dmrt6 gene is highly expressed in spermatocytes and is involved in spermatogenesis in tilapia.

The fox genes play important roles in various biological processes, including sexual development. In the present study, we isolated 65 fox genes, belonging to 18 subfamilies named A-R, from Nile tilapia through genome-wide screening. Twenty-four of them have two or three (foxm1) copies. Furthermore, 16, 25, 68, and 45 fox members were isolated from nematodes, protochordates, teleosts, and tetrapods, respectively. Phylogenetic analyses indicated fox gene family had undergone three expansions parallel to the three rounds of genome duplication during evolution. We also analyzed the clustered fox genes and found that apparent linkage duplication existed in teleosts, which further supported fish-specific genome duplication hypothesis. In addition, species- and lineage-specific duplication is another reason for fox gene family expansion. Based on the four pairs of XX and XY gonadal transcriptome data from four critical developmental stages, we analyzed the expression profile of all fox genes and identified sexually dimorphic fox genes at each stage. All fox genes were detected in gonads, with 15 of them at the background expression level (total read per kb per million reads, RPKM < 10), 29 at moderate expression level (10 < total RPKM < 100), and 21 at high expression level (total RPKM > 100). There are 27, 24, 28, and 9 sexually dimorphic fox genes at 5, 30, 90, and 180 days after hatching (dah), respectively. foxq1a, foxf1, foxr1, and foxr1 were identified as the most differentially expressed genes at each stage. foxl2 was characterized as XX-dominant gene, while foxd5, foxi3, foxn3, foxj1a, foxj3b, and foxo6b were characterized as XY-dominant genes. qPCR and in situ hybridization of foxh1 and foxj1a were performed to confirm the expression profiles and to validate the transcriptome data. Our results suggest that fox genes might play important roles in sex determination and gonadal development in teleosts.

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Zona pellucida (ZP) genes encode ZP glycoproteins which constitute the coat surrounding oocytes and early embryos. Genome-wide identification of ZP genes is still lacking in vertebrates, especially in fish species. Herein, we conducted bioinformatic analyses of the ZP genes of the Nile tilapia and other vertebrates. Totally 16, 9, 17, 27, 21, 20, 26, 19, 14,11, 24, 17, 9, 18, 8, 11, 9, 8, 5, and 4 ZP genes belonging to 5 subfamilies (ZPA, ZPB, ZPC, ZPD, and ZPAX) were found in the sea lamprey, elephant shark, coelacanth, spotted gar, zebrafish, medaka, stickleback, Nile tilapia, Amazon molly, platyfish, seahorse, Northern snakehead, cavefish, tetraodon, clawed frog, turtle, chicken, platypus, kangaroo rat, and human genomes, respectively. The expansion of ZP genes in basal vertebrates was mainly achieved by gene duplication of ZPB, ZPC, and ZPAX subfamilies, while the shrink of ZP gene number in viviparous mammals was achieved by keeping only one copy of the ZP genes in each subfamily or even secondary loss of some subfamilies. The number of ZP gene is related to the environment where the eggs are fertilized and the embryos develop in vertebrates. Transcriptomic analysis showed that 14 ZP genes were expressed in the ovary of Nile tilapia, while two (ZPB2b and ZPC2) were highly expressed in the liver. On the other hand, ZPB1a and ZPB2c were not found to be expressed in any tissue or at any developmental stage of the gonads examined. In the ovary, the expression of ZP genes started from 30 dah (days after hatching), significantly upregulated at 90 dah and maintained this level at 180 dah. The expression of ZPC2 in the liver and ZPC5-2 and ZPAX1 in the ovary was confirmed by in situ hybridization. The ovary- and liver-expressed ZP genes are expressed coordinately with oocyte growth in tilapia.

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