Activin and inhibin are both dimeric proteins sharing the same β subunits that belong to the TGF-β 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 β subunits (βAa, inhbaa; βAb, inhbab; and βB, inhbb) in zebrafish using CRISPR/Cas9. The loss of βAa/b but not βB 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 βA mutant together with aromatase (cyp19a1a) in the ovary. The single mutant of βAa/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 βAa/b mutant males showed normal spermatogenesis and fertility. As for activin βB 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.
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 complete arrest of follicle development at previtellogenic 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 rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-β signaling and endocytosis in the double mutant follicles. Intriguingly, the expression of inhibin/activin βAa 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 in the oocytes although the follicles could continue to grow beyond the size range of previtellogenic stage. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females, 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 vitellogenic follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. In summary, the present study provided comprehensive genetic evidence for the interaction of bmp15 pathways and the activin-inhibin system in regulating folliculogenesis, in particular E2 production from the follicle, Vtg biosynthesis in the liver and its update by the developing oocytes.
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 (♂) 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.
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