Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Regulation of spermatogenesis and reproductive capacity by Igf3 in tilapia.

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3-/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3-/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.

Establishment of a stem Leydig cell line capable of 11-ketotestosterone production.

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfrα), nestin and chicken ovalbumin upstream promoter transcription factor II (coup-tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs invitro and invivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis invitro, and provide an invaluable invitro model for investigating endocrine disruptors.

Transcription of the Sox30 Gene Is Positively Regulated by Dmrt1 in Nile Tilapia.

The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.

The First Total Synthesis of (±)-Methyl Salvianolate A Using a Convergent Strategy.

Herein, a convergent, practicable and first total synthesis of the natural product, (±)-methyl salvianolate A, is reported. The key features of the approach are the use of a Horner⁻Wadsworth⁻Emmons reaction and the protection of multiple hydroxyls using silyl protecting groups. The employment of the readily removable silyl protecting groups allows the synthesis of (±)-methyl salvianolate A and its derivatives on a reasonably large scale.

Cellulomonas macrotermitis sp. nov., a chitinolytic and cellulolytic bacterium isolated from the hindgut of a fungus-growing termite.

To investigate the symbiotic roles of the gut microbiota in the fungus-growing termite Macrotermes barneyi, a novel strain with chitinolytic and cellulolytic activity, designated strain an-chi-1T, was isolated from the hindgut of M. barneyi. Strain an-chi-1T grows optimally at 28-30 °C, pH 8.0 in PYG medium. On the basis of 16S rRNA gene sequence analysis, this isolate belongs to the genus Cellulomonas with high sequence similarity to Cellulomonas iranensis (99.4%), followed by Cellulomonas flavigena (98.4%), Cellulomonas phragmiteti (97.4%), Cellulomonas oligotrophica (97.2%) and Cellulomonas terrae (97.0%). The DNA-DNA relatedness between an-chi-1T and the type strains of C. iranensis and C. flavigena DSM20109T are 35.4% and 23.7%, respectively. The major cellular fatty acids are anteiso-C15:0 and C14:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylinositol dimannosides and one unidentified phospholipid. The cell-wall sugar is ribose. The peptidoglycan contains glutamic acid, aspartic acid and alanine. The DNA G+C content is 67.3 mol%. Based on its distinctive phenotypic, phylogenetic, and chemotaxonomic characteristics, an-chi-1T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas macrotermitis sp. nov. is proposed. The type strain is an-chi-1T (= JCM 31923T = CICC 24195T).

Steroid hormone-deprived sex reversal in cyp11a1 mutant XX tilapia experiences an ovary-like stage at molecular level.

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish.

Cortisol safeguards oogenesis by promoting follicular cell survival.

The role of glucocorticoids in oogenesis remains to be elucidated. cyp11c1 encodes the key enzyme involved in the synthesis of cortisol, the major glucocorticoid in teleosts. In our previous study, we mutated cyp11c1 in tilapia and analyzed its role in spermatogenesis. In this study, we analyzed its role in oogenesis. cyp11c1+/- XX tilapia showed normal ovarian morphology but poor egg quality, as indicated by the mortality of embryos before 3 d post fertilization, which could be partially rescued by the supplement of exogenous cortisol to the mother fish. Transcriptome analyses revealed reduced expression of maternal genes in the eggs of the cyp11c1+/- XX fish. The cyp11c1-/- females showed impaired vitellogenesis and arrested oogenesis due to significantly decreased serum cortisol. Further analyses revealed decreased serum E2 level and expression of amh, an important regulator of follicular cell development, and increased follicular cell apoptosis in the ovaries of cyp11c1-/- XX fish, which could be rescued by supplement of either exogenous cortisol or E2. Luciferase assays revealed a direct regulation of cortisol and E2 on amh transcription via GRs or ESRs. Taken together, our results demonstrate that cortisol safeguards oogenesis by promoting follicular cell survival probably via Amh signaling.

A Chromosome-Level Genome Assembly of Mozambique Tilapia (Oreochromis mossambicus) Reveals the Structure of Sex Determining Regions.

The Mozambique tilapia (Oreochromis mossambicus) is a fascinating taxon for evolutionary and ecological research. It is an important food fish and one of the most widely distributed tilapias. Because males grow faster than females, genetically male tilapia are preferred in aquaculture. However, studies of sex determination and sex control in O. mossambicus have been hindered by the limited characterization of the genome. To address this gap, we assembled a high-quality genome of O. mossambicus, using a combination of high coverage of Illumina and Nanopore reads, coupled with Hi-C and RNA-Seq data. Our genome assembly spans 1,007 Mb with a scaffold N50 of 11.38 Mb. We successfully anchored and oriented 98.6% of the genome on 22 linkage groups (LGs). Based on re-sequencing data for male and female fishes from three families, O. mossambicus segregates both an XY system on LG14 and a ZW system on LG3. The sex-patterned SNPs shared by two XY families narrowed the sex determining regions to ∼3 Mb on LG14. The shared sex-patterned SNPs included two deleterious missense mutations in ahnak and rhbdd1, indicating the possible roles of these two genes in sex determination. This annotated chromosome-level genome assembly and identification of sex determining regions represents a valuable resource to help understand the evolution of genetic sex determination in tilapias.

Differential expression patterns of the two paralogous Rec8 from Nile tilapia and their responsiveness to retinoic acid signaling.

REC8 (meiotic recombination protein 8) is an essential component of meiotic cohesion complexes. Interestingly, two paralogous rec8 genes happen to exist in the stra8 (stimulated by retinoic acid gene 8)-absent fishes but not in stra8-existing fishes. Stra8 is usually considered as the prerequirement during RA (retinoic acid)-mediated meiosis initiation in mammals. However, how RA triggers meiosis in the stra8-absent fishes just like Nile tilapia (Oreochromis niloticus) remains elusive. Here we characterized the two paralogous rec8 genes in Nile tilapia (Onrec8a and Onrec8b), and investigated their expression patterns and responsiveness to RA signaling by treatment of ex vivo testicular culture and promoter luciferase reporter assay. OnRec8a and OnRec8b share 36% identity to each other and are true orthologs of REC8. Their expression was predominantly restricted to meiotic germline cells with differential spatiotemporal patterns. During spermatogenesis, OnRec8b predominantly exhibited nuclear expression in spermatocytes from 60 dah (days after hatching), while OnRec8a exhibited cytoplasmic expression from 90 dah. During oogenesis, OnRec8a was expressed from 30 dah, while OnRec8b from 90 dah. Further study shows that RA signaling could upregulate the expression of both Onrec8a and Onrec8b. Collectively, our data implies that OnRec8a and OnRec8b might have differential function during meiosis and be involved in RA-mediated meiosis program.

Amh regulate female folliculogenesis and fertility in a dose-dependent manner through Amhr2 in Nile tilapia.

In the present study, Amh was found to be abundantly expressed in the granulosa cells of the primary growth follicles, and Amhr2 in the granulosa cells, oogonia and phase I oocytes in tilapia by immunohistochemistry. In addition, Amh and Amhr2 were also found to be expressed in the brain and pituitary. Heterozygous mutation of either amh or amhr2 resulted in increased primary growth follicles and decreased fertility, and homozygous mutation resulted in hypertrophic ovaries with significantly increased primary follicles and failed transition from primary to vitellogenic follicles. Expression of gnrh3 in the brain, fsh and lh in the pituitary and serum E2 concentration were significantly decreased in both mutants. Significantly increased apoptosis of follicle cells was observed in both mutants. However, administration of E2 failed to rescue the folliculogenesis defects of the mutants. Our results suggested that Amh acts in a dose-dependent manner by binding Amhr2 in tilapia.

Loss of Cyp11c1 causes delayed spermatogenesis due to the absence of 11-ketotestosterone.

The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11ß-hydroxylase, encoded by cyp11c1, is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a smaller efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia.

High Efficiency Targeting of Non-coding Sequences Using CRISPR/Cas9 System in Tilapia.

The CRISPR/Cas9 has been successfully applied for disruption of protein coding sequences in a variety of organisms. The majority of the animal genome is actually non-coding sequences, which are key regulators associated with various biological processes. In this study, to understand the biological significance of these sequences, we used one or dual gRNA guided Cas9 nuclease to achieve specific deletion of non-coding sequences including microRNA and 3' untranslated region (UTR) in tilapia, which is an important fish for studying sex determination and evolution. Co-injection of fertilized eggs with single gRNA targeting seed region of miRNA and Cas9 mRNA resulted in indel mutations. Further, co-injection of fertilized eggs with dual gRNAs and Cas9 mRNA led to the removal of the fragment between the two target loci, yielding maximum efficiency of 11%. This highest genomic deletion efficiency was further improved up to 19% using short ssDNA as a donor. The deletions can be transmitted through the germline to the next generation at average efficiency of 8.7%. Cas9-vasa 3'-UTR was used to increase the efficiency of germline transmission of non-coding sequence deletion up to 14.9%. In addition, the 3'-UTR of the vasa gene was successfully deleted by dual gRNAs. Deletion of vasa 3'-UTR resulted in low expression level of vasa mRNA in the gonad when compared with the control. To summarize, the improved CRISPR/Cas9 system provided a powerful platform that can assist to easily generate desirable non-coding sequences mutants in non-model fish tilapia to discovery their functions.