Journal: PLOS Genetics

Article Title: Rescue of bmp15 deficiency in zebrafish by mutation of inha reveals mechanisms of BMP15 regulation of folliculogenesis

doi: 10.1371/journal.pgen.1010954

Figure Lengend Snippet: (A) Stage-matching ovarian samples collected at 60 dpf. Both PG and PV follicles were present in bmp15+/- and bmp15-/- ovaries. (B) Expression of selected genes at PG and PV stages in bmp15+/- and bmp15-/- fish. The expression of cyp19a1a and inhbaa were dramatically reduced in PV follicles of bmp15-/- fish. cyp19a1a , ovarian aromatase; fshr and lhcgr , FSH and LH receptors; inhbaa , inhbab and inhbb , activin/inhibin β subunits; inha , inhibin α subunit; fsta and fstb , follistatins. The relative mRNA levels were determined by real-time qPCR, normalized to the housekeeping gene ef1a , and expressed as fold change compared with the levels at the PG stage of the control fish. The values are expressed as mean ± SEM (n = 5) and analyzed by ANOVA followed by the Tukey HSD for multiple comparisons (*** P < 0.001; n.s., no significance). (C) Levels of E2 and Vtg in the serum of bmp15+/- and bmp15-/- females at 120 dpf. The E2 and Vtg contents of bmp15-/- were both significantly lower than those in the control. The values are expressed as mean ± SEM (n = 5) and analyzed by t-test (** P < 0.01; *** P < 0.001).

Article Snippet: Both Cas9 mRNA (300 ng/μL) and sgRNA (75 ng/μL) were co-microinjected (4.6 nL) into zebrafish embryos at one- or two-cell-stage using the Drummond Nanoject injector (Drummond Scientific, Broomall, PA).

Techniques: Expressing, Control

Journal: PLOS Genetics

Article Title: Rescue of bmp15 deficiency in zebrafish by mutation of inha reveals mechanisms of BMP15 regulation of folliculogenesis

doi: 10.1371/journal.pgen.1010954

Figure Lengend Snippet: (A) Morphology and histology of the double mutant gonads ( bmp15-/-;inha-/- ) after fadrozole treatment. The females were reared in 10 L tanks from 80 to 100 dpf with dried powder feed containing fadrozole (200 μg/g diet) at 10% (W/W) of fish body weight per day. The resumption of follicle development to vitellogenic stage in the double mutants was completely abolished by treatment with fadrozole. The numbers shown in each sample indicate total number of fish sampled (lower) and the number of fish that showed the same phenotype (upper). (B) Expression of cyp19a1a in the PG and PV follicles of the control and mutants after E2 and fadrozole treatments. (C) Expression of vtg1 and vtg3 in the liver of the control and mutants after E2 and fadrozole treatments at 120 dpf. The relative mRNA levels were determined by real-time qPCR, normalized to the housekeeping gene ef1a , and expressed as fold change compared with the levels in the control fish. (D) Levels of E2 and Vtg in the serum of the control and mutants after E2 and fadrozole treatments. The values are expressed as mean ± SEM (n ≥ 3) and analyzed by ANOVA followed by the Tukey HSD for multiple comparisons. Different letters indicate statistical significance (P < 0.05).

Article Snippet: Both Cas9 mRNA (300 ng/μL) and sgRNA (75 ng/μL) were co-microinjected (4.6 nL) into zebrafish embryos at one- or two-cell-stage using the Drummond Nanoject injector (Drummond Scientific, Broomall, PA).

Techniques: Mutagenesis, Expressing, Control

Journal: PLOS Genetics

Article Title: Rescue of bmp15 deficiency in zebrafish by mutation of inha reveals mechanisms of BMP15 regulation of folliculogenesis

doi: 10.1371/journal.pgen.1010954

Figure Lengend Snippet: (A) Expression of ovarian genes in the PG follicles ( cyp19a1a , fshr , lrp1aa and lrp2a ). (B) Expression of Vtg genes in the liver ( vtg1-7 ). (C) Expression of estrogen receptors in the liver ( esr1 , esr2a and esr2b ). (D) Expression of gonadotropins in the pituitary ( fshb and lhb ). The relative mRNA levels were determined by real-time qPCR, normalized to the housekeeping gene ef1a and presented as the fold change compared with the control fish. Data were analyzed by ANOVA followed by the Tukey HSD for multiple comparisons (* P < 0.05; ** P < 0.01; *** P < 0.001; n ≥ 3). (E) In situ hybridization on the expression of fshb (red) and lhb (green) in the pituitary of different genotypes. DAPI stains for nuclei (blue).

Article Snippet: Both Cas9 mRNA (300 ng/μL) and sgRNA (75 ng/μL) were co-microinjected (4.6 nL) into zebrafish embryos at one- or two-cell-stage using the Drummond Nanoject injector (Drummond Scientific, Broomall, PA).

Techniques: Expressing, Control, In Situ Hybridization