Rescue of bmp15 deficiency in zebrafish by mutation of inha reveals mechanisms of BMP15 regulation of folliculogenesis.

As an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by a complete arrest of follicle development at previtellogenic (PV) stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal in bmp15 mutant could be partially rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic (MV) stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-ß signaling and endocytosis in the double mutant follicles. Interestingly, the expression of inhibin/activin ßAa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females (bmp15-/-), returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the arrested follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. The loss of inhbaa also caused a reduction of Vtg receptor-like molecules (e.g., lrp1ab and lrp2a). In summary, the present study provided comprehensive genetic evidence that Bmp15 acts together with the activin-inhibin system in the follicle to control E2 production from the follicle, Vtg biosynthesis in the liver and its uptake by the developing oocytes.

Genetic analysis of activin/inhibin ß subunits in zebrafish development and reproduction.

Activin and inhibin are both dimeric proteins sharing the same ß subunits that belong to the TGF-ß superfamily. They are well known for stimulating and inhibiting pituitary FSH secretion, respectively, in mammals. In addition, activin also acts as a mesoderm-inducing factor in frogs. However, their functions in development and reproduction of other species are poorly defined. In this study, we disrupted all three activin/inhibin ß subunits (ßAa, inhbaa; ßAb, inhbab; and ßB, inhbb) in zebrafish using CRISPR/Cas9. The loss of ßAa/b but not ßB led to a high mortality rate in the post-hatching stage. Surprisingly, the expression of fshb but not lhb in the pituitary increased in the female ßA mutant together with aromatase (cyp19a1a) in the ovary. The single mutant of ßAa/b showed normal folliculogenesis in young females; however, their double mutant (inhbaa-/-;inhbab-/-) showed delayed follicle activation, granulosa cell hypertrophy, stromal cell accumulation and tissue fibrosis. The ovary of inhbaa-/- deteriorated progressively after 180 dpf with reduced fecundity and the folliculogenesis ceased completely around 540 dpf. In addition, tumor- or cyst-like tissues started to appear in the inhbaa-/- ovary after about one year. In contrast to females, activin ßAa/b mutant males showed normal spermatogenesis and fertility. As for activin ßB subunit, the inhbb-/- mutant exhibited normal folliculogenesis, spermatogenesis and fertility in both sexes; however, the fecundity of mutant females decreased dramatically at 270 dpf with accumulation of early follicles. In summary, the activin-inhibin system plays an indispensable role in fish reproduction, in particular folliculogenesis and ovarian homeostasis.

RNase1 can modulate gut microbiota and metabolome after Aeromonas hydrophila infection in blunt snout bream.

Pancreatic ribonuclease (RNase1) of Megalobrama amblycephala exhibits both antimicrobial and digestive activity. The gut microbiome improve the digestion and metabolic capacity and enhance the functioning of the immune system of the host against pathogenic bacteria. In this study, we aimed to assess the protective effect of RNase1 on Aeromonas hydrophila-induced inflammation and intestinal microbial metabolism. Megalobrama amblycephala were randomly divided into three groups: control (injected PBS), infection (A. hydrophila-injected), and treatment group (RNase1 pretreatment 24 h before the A. hydrophila injection). The morphological symptoms were significantly alleviated by RNase1. RNase1 reshaped the perturbed gut microbiota by upregulating Proteobacteria and Vibrio richness and downregulating Firmicutes, Chlamydiae, Bacillus, and Gemmobacter richness. The lysophosphatidylcholine, (±) 17 HETE, D- (+) -cellobiose, and PC (20:5) in the treatment group were restored by RNase 1 protein treatment to the level of the control group. In the treatment group, phospholipid metabolism, fatty acid metabolism, glucose metabolism and lipid metabolism were different from the control and infection groups. The proinflammatory factors concentration in intestinal samples significantly increased after A. hydrophila infection. Our results revealed that RNase1 plays an important role in resistance to pathogen invasion, reducing inflammation, and improving intestinal function, thus inhibiting the occurrence of disease.

Ribonuclease1 contributes to the antibacterial response and immune defense in blunt snout bream (Megalobrama amblycephala).

Ribonuclease 1 (RNase1) is a vertebrate-specific enzyme that mainly performs digestive activity in herbivorous mammals. Here we used bacterial viability assays to explore its antimicrobial activity in blunt snout bream (Megalobrama amblycephala). The results showed that Ma-RNase1 rapidly killed Gram-negative and Gram-positive bacteria at micromolar concentrations. Ma-RNase1 increased the permeability of bacterial outer and inner membranes, thus reducing the integrity of bacterial cell wall and membrane. Moreover, Ma-RNase1 effectively counteracted the tissue damage and apoptosis caused by Aeromonas hydrophila infection. Quantitative real-time PCR and immunoblot analysis indicated that RNase1 mRNA and protein were up-regulated in the kidney and gut during infection. Furthermore, A. hydrophila infection significantly induced Tnf-α and Il-1ß mRNA expression in liver, but not in the RNase1 pre-treatment group. In addition, a significant increase in the expression of immune-related genes (Nf-κb and Tlr4) was found in liver, kidney and gut of A. hydrophila-infected fish, while a decrease in Myd88 and Tlr4 levels was found in liver, spleen, kidney and gut in the group pre-treated with RNase1. Collectively, these data suggest that Ma-RNase1 has antimicrobial function both in vitro and in vivo, and contributes to the protective effect and immune defense of blunt snout bream.

Anatomical structure, and expression of CCL4 and CCL13-like during the development of maxillary barbel in Paramisgurnus dabryanus.

Paramisgurnus dabryanus is one of the most economically important fishes in China. Barbels are an essential sensory organ for the food-seeking ability of teleost fish. However, the anatomical structure of the maxillary barbels of P. dabryanus and the molecular basis of their development are unknown. We investigated the anatomical structure of the barbel, and gene expression patterns of two chemokine C-C motif ligands: CCL4 and CCL13-like during the maxillary barbel development using Masson Trichrome staining, light and electron microscopy, and qPCR. Anatomically, the maxillary barbel of P. dabryanus contains taste buds, melanophores, collagen fibers, connective tissue, smooth muscles, nerve bundles, and blood vessels, but does not have skeletal muscles or a skeleton rod. The expression of CCL4 and CCL13-like was weak or non-existent in the early phases of development, but high at the last two studied time-points: 192- and 216-h post-hatching. Results indicated that CCL4 and CCL13-like were related to the development of the maxillary barbel.

RNase1 alleviates the Aeromonas hydrophila-induced oxidative stress in blunt snout bream.

RNase1 is an enzyme important in host defense in vertebrates where it degrades the RNA of bacteria and viruses. We evaluated the effect of RNase1 on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into four groups: a blank group (none-treated M. amblycephala), a control group (injected PBS), a challenge group (A. hydrophila-injected) and a treatment group (pre-treated with RNase1 24 h before the A. hydrophila injection), and we collected five tissues of each group. Then we recorded changes in the levels of glutathione (GSH), oxidized glutathione (GSSG), hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and lysozyme; and the relative mRNA expression of catalase (CAT), selenium-dependent glutathione peroxidase (GPx), Cu/Superoxide dismutase (Cu/Zn-SOD), glutamate-cysteine ligase (GCLC), glutathione reductase (GR) and nuclear factor erythroid 2-related factor 2 (Nrf2) for four groups. The expression of six genes was highest in liver and blood of the blank group. It was significantly higher in the gut of the treatment group (compared to control and challenge groups) 12 h after the infection. The treatment group exhibited a significant increase in GSH, SOD and CAT activity, and a decrease in GSSG, MDA and lysozyme content (compared to the control and challenge groups) 6 and 12 h after infection. These results suggest that supplementation with RNase1 protein can enhance resistance against A. hydrophila infections in M. amblycephala.

Differential Expression of Six Rnase2 and Three Rnase3 Paralogs Identified in Blunt Snout Bream in Response to Aeromonas hydrophila Infection.

Ribonucleases (Rnases)2 and Rnase3 belong to the ribonuclease A (RnaseA) superfamily. Apart from their role in molecular evolutionary and functional biological studies, these genes have also been studied in the context of defense against pathogen infection in mammals. However, expression patterns, structures and response to bacterial infection of the two genes in blunt snout bream (Megalobrama amblycephala) remain unknown. In this study, we identified multiple copies of Rnase2 (six) and Rnase3 (three) in the M. amblycephala genome. The nine genes all possess characteristics typical of the RnaseA superfamily. No expression was detected in the early developmental stages, while a weak expression was observed at 120 and 140 h post-fertilization (hpf) for Rnase2b, Rnase2c, Rnase2e and Rnase3a, suggesting that only three copies of Rnase2 and one of Rnase3 are expressed. Interestingly, only Rnase2e was up-regulated in the kidney of M. amblycephala after Aeromonas hydrophila infection, while Rnase3a was significantly up-regulated in liver, gut and blood after the infection. We conclude that the paralogs of Rnase3 are more susceptible to A. hydrophila infection than Rnase2. These results indicate that different Rnase2 and Rnase3 paralogs suggest a role in the innate immune response of M. amblycephala to bacterial infection.

The complete mitochondrial genome of the hybrid of Megalobrama amblycephala (♀) × Megalobrama skolkovii (♂).

The complete mitochondrial genome of the hybrid of Megalobrama amblycephala (♀) × Megalobrama skolkovii (♂) was characterized first in this study. The total length of the genome was identical to the female parent as 16 623 bp, and the overall base composition was 31.23% A, 24.69% T, 27.89% C, and 16.19% G, with a slight A + T bias. It contained 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions (the control region and the origin of the light-strand replication). This study discovered the 99.88% sequence identity between the hybrid and its female parent, which confirmed the maternal inheritance pattern followed by the mitochondrial genome of the hybrid. However, the sequence alignment of mitochondrial genomes between the hybrid and its female parent revealed a total of 20 variable sites in 10 genes or regions, especially 4 sense mutations in 2 PCGs (COX1 and ATPase6). The complete mitochondrial genome sequence of this hybrid bream may provide an important dataset for further study in mitochondrial inheritance mechanism.