Fig 1: CCK regulates the synthesis and function of pituitary FSH and LH in zebrafish.a Representative immunohistochemical staining of LH and FSH proteins in the pituitary of WT, cck1−/−;cck2−/−, and cckbrb−/− zebrafish. Representative images from three independent experiments with similar results. b Relative mRNA expression of lhb and fshb in pituitary tissue from different knockout models compared to WT. Data are derived from n = 3 independent biological replicates, where each replicate consists of a pool of 3 pituitaries. c Single-cell RNA sequencing (scRNA-seq) visualization of zebrafish pituitary cell clusters. d Sub-clustering and trajectory analysis of the gonadotrope cell population. e, f Regulation of fshb expression. e Pituitary fshb mRNA levels measured 24 hours after intraperitoneal (i.p.) injection of CCK1 or CCK2. Data represent n = 4 independent biological replicates, each consisting of a pool of 4 pituitaries. f fshb mRNA levels in primary pituitary cells treated with CCK1 or CCK2 for 24 hours (n = 4). g, h, Analysis of the upstream hypothalamic gnrh3 response. g Hypothalamic gnrh3 expression in CCK ligand and receptor knockout models (n = 6). h gnrh3 mRNA levels in hypothalamic cells treated with CCK1 or CCK2 for 3 h and 24 h (n = 4). i, j Regulation of lhb expression. i lhb mRNA levels in primary pituitary cells treated with CCK1 or CCK2 for 24 h. Data represent n = 4 independent biological replicates, each consisting of a pool of 4 pituitaries. j Pituitary lhb mRNA levels measured 24 hours after i.p. injection of CCK1 or CCK2. Data represent n = 4 independent biological replicates, each consisting of a pool of 3 pituitaries. k Functional assessment of ovulation success rate (%) in female zebrafish 24 hours after i.p. injection of LH or CCK (n = 14 biologically independent animals from 3 independent experiments). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple-comparisons test for (b, e–k). Data are presented as mean ± s.e.m.
Fig 2: CCK signaling regulates germ cell meiosis and gonadal somatic cell survival.a Immunofluorescence showing the expression of VASA and SYCP3 in gonadal tissue of 14 dpf WT and cckbrb−/− zebrafish. b RNA-seq analysis of testis tissue from WT and cck1−/−;cck2−/− zebrafish. Volcano plot (left) and gene lists (right) display differentially expressed genes related to meiosis and spermiogenesis. Differential expression analysis was performed as described in the Methods. c qPCR analysis of meiosis-related gene expression in testes of WT and cck1−/−;cck2−/− zebrafish. Data are presented as mean ± s.d. (n = 4). Statistical significance was determined by two-sided unpaired Student’s t test. d VASA and SYCP3 co-staining in gonadal sections of 14 dpf cck1−/−;cck2−/− larvae following exogenous CCK immersion treatment starting from fertilization. e Co-staining of VASA, TUNEL, and SOX9a in testes of 60 dpf zebrafish. f TUNEL and VASA staining in 14 dpf gonads of WT, fshb−/−, lhb−/−;fshb−/−, and cckbrb−/− zebrafish. g, h Analysis of cckbrb−/−;tp53−/− zebrafish. g Immunofluorescence of SYCP3 and VASA in testes at 45 dpf. h Gross morphology and histological analysis (H&E) of gonads. i Schematic diagram depicting the stage-specific regulatory network of CCK signaling during gonadal development. The model illustrates CCK involvement in primordial germ cell (PGC) proliferation during early formation, the maintenance of somatic cell integrity (via apoptosis inhibition) and meiotic initiation during differentiation, and the regulation of pituitary FSH and LH synthesis (involving both direct pituitary action and indirect hypothalamic gnrh3 modulation) to support gametogenesis and ovulation. Created in BioRender. Li, H. (2026) https://biorender.com/o68vxgm. For (a, d–h), representative images from three independent experiments with similar results are shown.
Fig 3: Impact of CCK signaling on primordial germ cell (PGC) proliferation and migration in zebrafish.a, b Analysis of PGC number and distribution in zebrafish embryos at 24 hours post-fertilization (hpf). PGCs were visualized using Piwi1 immunofluorescence staining. a Representative images and quantification of PGCs in wild-type (WT), cckbrb−/−, and cck1−/−;cck2−/− embryos. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test. ns indicates no significant difference where applicable. Data are presented as mean ± s.e.m. b Representative images and quantification of PGCs in fshb+/+, fshb−/−, lhb+/+;fshb+/+ and lhb−/−;fshb−/− embryos. Statistical significance was determined by two-sided unpaired Student’s t test. ns indicates no significant difference where applicable. Data are presented as mean ± s.e.m. (n = 10 embryos per group). c Temporal expression profiles of CCK ligands and receptors during early development. Relative mRNA levels of ligands (cck1, cck2) and receptors (cckar, cckbra, cckbrb) were quantified by qPCR from 1 to 7 days post-fertilization (dpf). Data are presented as mean ± s.e.m. for n = 3 independent replicates. Each replicate represents a pooled sample of 20–30 embryos or larvae. d Whole-mount immunofluorescence co-staining of VASA (germ cell marker) and CCK in zebrafish larvae at 2 dpf and 5 dpf. Merged images show the spatial relationship between CCK-expressing cells and migrating PGCs. e Immunofluorescence of CCK and VASA in 14 dpf gonads reveals CCK expression in somatic cells surrounding germ cells. f Immunofluorescence co-expression analysis of germ cells (VASA) and mitotic cells (Histone H3) in gonadal tissue of WT, fshb−/−, lhb−/−;fshb−/−, and cckbrb−/− zebrafish at 14 dpf. For (d, f), representative images from three independent experiments with similar results are shown.
Fig 4: Generation and phenotypic characterization of the cckbrb knockout zebrafish model.a Ligand-receptor binding analysis. Structural prediction of zebrafish CCK interacting with its receptors (CCKAR, CCKBRA, and CCKBRB) via AlphaFold3 and functional validation using a dual-luciferase reporter assay (n = 4). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple-comparisons test. ns indicates no significant difference where applicable. Data are presented as mean ± s.e.m. b Gonadal morphology and histology in 5-month-old cckbrb+/+ and cckbrb−/− zebrafish. Quantification includes the distribution of gonadal cell stages (n = 7), sex ratio, and gonadosomatic index (GSI) (n = 15). Statistical significance was determined by two-sided unpaired Student’s t test. ns indicates no significant difference where applicable. Data are presented as mean ± s.e.m. c VASA and SYCP3 immunofluorescence in testes of cckbrb+/+ and cckbrb−/− zebrafish. Representative images from three independent experiments with similar results are shown. d Analysis of secondary sexual characteristics (SSC) in 4-month-old zebrafish. Representative images comparing the anal fin (AF), caudal fin (CF), and pectoral fin (PF) phenotypes in wild-type, cck1−/−;cck2−/−, and cckbrb−/− zebrafish. e Reproductive behavior analysis assessing courtship frequency in cckbrb−/− zebrafish (n = 24 independent pairings). Statistical significance was determined by two-sided unpaired Student’s t test. In the box plots, the center line indicates the median, the box bounds indicate the first and third quartiles, and the whiskers indicate the minimum and maximum values.
Supplier Page from Cosmo Bio USA for Cholecystokinin (CCK) ELISA Kit (fish)