Cystic proliferation of germline stem cells is necessary to reproductive success and normal mating behavior in medaka
- Instituto Tecnológico de Chascomús, INTECH (CONICET-UNSAM), Argentina
- Reproductive and Molecular Biology Group, Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Brazil
- Salmonid Experimental Station at Campos do Jordão, UPD-CJ, Sao Paulo Fisheries Institute (APTA/SAA), Brazil
Figures
Figure 1 with 1 supplement
ndrg1b is down-regulated during cystic proliferation in gonadal development.
Phylogenetic tree showing the relationships among the ndrg family members in medaka (A), obtained using the neighbor-joining method and a bootstrap test (MEGA 7.0 software). The scale beneath the …
Figure 1—figure supplement 1
TGF-β is not involved in the cystic proliferation during early gonadal development.
Schematic representation of the experimental procedure adopted for TGF-β inhibitor (A83-01) treatment (A). Proposed hypothesis (B). Total number of embryonic germline stem cells in XX and XY groups …
Figure 2 with 4 supplements
The mutation of ndrg1b affects gonadal development at stage 39.
Schematic representation of the experimental procedure adopted to analyze the effect of ndrg1b loss on germ cell number and proliferation (A: anterior, P: posterior, D: dorsal; and V: ventral) (A). …
Figure 2—figure supplement 1
CRISPR/Cas9: sg1_ndrg1b design, heteroduplex mobility assay (HMA), efficiency, and potential off-targets.
Schematic representation of the genomic structure of ndrg1b. Coding exon regions are shown as blue solid boxes, the targeting sequence of sg1_ndrg1b is indicated by the cyan box, and NGG protospacer …
Figure 2—figure supplement 2
Corroboration of the specificity of CRISPR/Cas9 methodology to mutate ndrg1b with a second RNA guide (sg2_ndrg1b).
Schematic representation of the genomic structure of ndrg1b. Coding exon regions are shown as blue solid boxes. The targeting sequence of sg2_ndrg1b is indicated by the orange box and NGG …
Figure 2—figure supplement 3
Identification of ndrg1b KO fish in an F2 trial.
Schematic representation of the procedure to obtain an F2 family obtained by mating F1 fish harboring the Δ31 mutation with each other (A). Table indicates the obtained proportion of heterozygous …
Figure 2—figure supplement 4
Apoptosis of embryonic germline stem cells (EGSCs) at stage 39.
Percentage of TUNEL-positive EGSCs at stage 39 was determined using 10 sagittal sections per individual (n = 4) (A). Fluorescent images of gonads from female or male embryos injected with cas9 (B) …
Figure 3
The mutation of ndrg1b affects gonadal development at 20 days post hatching (dph).
Schematic representation of the experimental procedure adopted to analyze the effect of ndrg1b mutation on gonadal histology at 20 dph (D: dorsal, V: ventral) (A). Histological transverse sections …
Figure 4 with 1 supplement
Mutation of ndrg1b alters the gametogenesis and reproduction success of both sexes in adults.
Schematic representation of the experimental procedure adopted to analyze the effect of ndrg1b loss of function in the reproductive success at 80 days post hatching (dph) (A). Reproductive success …
Figure 4—figure supplement 1
F1 reproductive success was evaluated by crossing the wild-type (wt) males with F1 females (cyan full), wt females with F1 males (cyan empty), and as a control, wt females with wt males (gray).
For all F1 crossings, we quantified the total number of eggs spawned (A), percentage of fertilization (B), and percentage of hatching (C) of the total eggs spawned. p-Value is indicated when between …
Figure 5 with 2 supplements
Reduction of cystic proliferation in ndrg1b mutants decreases male sexual behavior, with a decrease of mating vigor.
Schematic representation of the experimental procedure adopted to analyze reproductive behavior for 5 days; five couples per group were analyzed. (A) Couples of wild-type (wt) individuals (gray) or s…
Figure 5—figure supplement 1
ndrg1b mutation did not alter the 11-ketotestosterone (11-KT) levels in medaka adults.
Quantification of whole blood 11-KT levels in male adults (A). Wild-type male (represented by gray full bars), and sgN1b male (represented by cyan full bars) (B). n = 6 per group. p-Values are …
Figure 5—figure supplement 2
ndrg1b is expressed only in presumptive A-type spermatogonia (SG) from the testis.
Transcript abundance levels of ndrg1b in male adult brain and testis (A). Quantification was performed using the 2-ΔΔCt method, and ndrg1b values were normalized to rpl7. Brain and testis are …
Author response image 1
Additional files
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Supplementary file 1
(A) Primers sequences, ENSEMBL accession numbers and respective references of each gene were added. (B) Sex ratio of both sexes embryos injected with cas9 (wildtype) and the sgNb1 (cas9+sg1_ndrg1b).
- https://cdn.elifesciences.org/articles/62757/elife-62757-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62757/elife-62757-transrepform-v2.docx
