Effect of CCK-R loss-of-function mutation on gonadal development.

(A) Expression of CCKR and two identified GnRH receptors in LH and FSH cells. Expression data were taken from a transcriptome of sorted pituitary cells of transgenic Nile tilapia (Oreochromis niloticus), expressing RFP in LH cells and GFP in FSH cells, previously obtained by Hollander-Cohen et al (1). Each dot represents a FACS fraction from a bulk of 20 pituitaries (n=8, 4 groups of males and 4 groups of females). The expression of each gene in each cell type is normalised to its expression in non-gonadotroph pituitary cells. While GnRHR3 is the dominant receptor in LH cells, CCKR has a significantly higher expression in FSH cells (two-way ANOVA, * p<0.05,). (B) RNA expression of CCKR (white) identified by hybridization chain reaction (HCR) in transgenic zebrafish pituitaries expressing RFP in LH cells (magenta) and GFP in FSH cells (green) shows specific expression predominanly on FSH cells. (C) Immunohistochemical staining of CCK (white) in transgenic zebrafish expressing GFP in FSH cells (left panel) or GFP in GnRH neurons (right panel). The CCK neuronal axons that innervate the pituitary gland are located adjacent to GnRH axons and in very close proximity to FSH cells. (D) H&E staining of body cross-sections (dorsoventral axis) of adult WT, heterozygous (wt/ CCKR-+12/+7/-1), and KO zebrafish (CCKR-+12/+7/-1/ CCKR-+12/+7/-1). An inset of the red square in each image on the right displays a magnified view of the gonad. All KO zebrafish exhibit small undeveloped male gonads. On the top right of each panel is the gender distribution for each genotype, while a similar distribution of males and females was identified in the WT and heterozygous; the KO zebrafish were only males. (E) Gonad areas of mutant zebrafish. All KO zebrafish exhibit a small gonad compared to the WT and the heterozygous (n((+/+), (+/-), (-/-))=10/6/17,one way ANOVA,, ****p<0.0001). (F) The distribution of cell types in the gonads of WT, heterozygous and KO zebrafish. CCKR LOF affects the gonad cell distribution and significantly reduces the maturation of the cells to spermatozoa. (n((+/+),(+/-), (-/-))=10/6/17, two-way ANOVA, * p<0.05, ** p<0.001, *** p<0.0001,****p<0.00001). (G) Gonadotroph mRNA expression in the pituitaries of the three genotypes revealed a significant decrease in both LH and FSH beta subunit expression in the KO fish(n((+/+),(+/-), (-/-))=9/8/10, one-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001).

LH and FSH cells exhibit distinct spontaneous activity patterns in vivo.

(A) Triple transgenic zebrafish were generated by crossing transgenic zebrafish expressing GFP in FSH cells to zebrafish lines expressing R-CaMP2 in LH and FSH cells. A confocal image of the pituitary shows RCaMP2 expression in both cell types and GFP expression in FSH cells. (B) A diagram describing the setup of the in vivo experiments. The dissected zebrafish were placed in a chamber with a constant flow of water to the gills and imaged in an upright two-photon microscopy. (C) A representative image of in vivo calcium activity (see movie. S2). On the top left is a merged image depicting FSH cells in green and LH cells in magenta. The other top panels show sequential calcium imaging, which reveals calcium rise in all LH cells followed by calcium rise in FSH cells (marked by white arrows). The bottom panels show the calcium of LH and FSH cells in two different imaged pituitaries, one where only FSH cells were active (Fish 1) and another where both cell types were active (Fish 2, traces ΔF/F, see supplementary fig. 1a for heatmap of the calcium traces). (D) The properties of spontaneous calcium transients (ΔF/F) in LH cells and FSH cells in three males and one female. Means of peak amplitude and duration differed between LH and FSH cells (unpaired t-test, ** p<0.001). Analysis was performed using pCLAMP 11. (E) Left: Cross correlation analysis between active ROI to the rest of the cell. The color-coded data points are superimposed on the imaged cells and represent the maximum cross-correlation coefficient between a calcium trace of a region of interest (ROI) and that of the rest of the cells in the same population. Right: is a matrix of maximum cross-correlation coefficient values between all the cells. All LH cells exhibited highly correlated calcium activity. In comparison, the activity of FSH cells was less synchronized to different extents (e.g. Fish 1 vs Fish 2). (F) Summary of the mean max cross-correlation coefficient values of calcium traces in each cell population of repeated in vivo calcium imaging assays (n (calcium sessions) =16, see Supplementary Fig. 1b for all measurements). The values for LH cells are significantly higher and more uniform than those of FSH cells (unpaired t-test, *** p<0.0001).

GnRH induces a synchronized increase of calcium in all LH cells.

(A)Top: Image of a dissected head with pituitary exposed from the ventral side of the fish used for the ex vivo assays (OB, olfactory bulb; OC, optic chiasm; PIT, pituitary; TH, thalamus; MO, medulla oblongata). Bottom: A diagram describing the ex vivo setup with a constant flow of artificial cerebrospinal fluid (ACSF), a side tube to inject stimuli, and a collecting tube. (B) A representative analysis of basal calcium activity of LH and FSH cells. The left panel is a heatmap of calcium traces (ΔF/F), where each line represents a cell, with the mean calcium trace on top, the separated line at the bottom of each heatmap is the calcium trace of the chosen ROI. The color-coded data points on the right are superimposed on the imaged cells and represent the maximum cross-correlation coefficient between a calcium trace of an active chosen ROI to those of the rest of the cells in the same population, matrix on the right represent the maximum cross-correlation coefficient values between all the cells. LH cells exhibited short calcium rises that were correlated between small groups of neighbouring cells (see supplementary fig. 3), whereas short, unorganized calcium transients were observed in FSH cells (see supplementary fig. 2 for additional cell activity parameters). (C) An analysis of calcium response to GnRH stimulation in two representative imaging sessions. In all LH cells, a significant calcium rise was highly correlated. In 5/10 fish, a similar response to the stimulus was seen in FSH cells (see supplementary fig. 4 for detailed coefficient values distribution in each fish), albeit with lower calcium intensities and coefficient values (e.g., Fish 1), whereas in the other 5 fish FSH cells did not respond at all (e.g., Fish 2). (D) The mean of Max cross-correlation coefficient values in each cell type under each treatment (see supplementary fig. 4 for detailed coefficient values distribution in each fish) reveal that only LH cells are significantly affected by GnRH stimuli (n=10, 3 males, 7 females, one-way ANOVA, **** p<0.0001). (E) The percentage of cells responsive to GnRH stimulus (i.e., coefficient values higher than the 80 percentiles of basal values) is significantly higher in LH cells compared to FSH cells. Each dot represents one fish (n=10, 3 males, 7 females, unpaired t-test, *** p<0.001).

FSH cells are directly stimulated by CCK.

(A) Example of calcium analysis of FSH and LH cells during CCK stimulation: fish1 with only FSH cells responding, and fish2 with FSH and LH cells responding. For each fish the left panels are a heatmaps of calcium traces (ΔF/F), where each line represents a cell. On top of each heatmap is a graph showing the mean calcium trace, the separated line at the bottom of the heat map is the calcium trace of the chosen ROI. On the right are color-coded data points that are superimposed on the imaged cells, showing the maximum cross-correlation coefficient between a calcium trace of a chosen active ROI and those of the rest of the cells in the same population, next to it is a matrix of max cross correlation coefficients between all the cells. A high calcium rise (2.5 ΔF/F) was observed in FSH cells, while LH cells in some zebrafish responded with a very low amplitude. (B) The mean of max cross-correlation coefficient values in each cell type reveals that only FSH cells are significantly affected by CCK stimulation (n=7, 4 males, 3 females, one-way ANOVA, **** p<0.0001, see supplementary fig. 4 for detailed coefficient values distribution in each fish). (C) The percentage of active cells (i.e., a coefficient value higher than the 80 percentiles of basal levels) during CCK stimulation is significantly higher in FSH cells compared to LH cells. While in all the fish, 96%-100% of the FSH cells responded to CCK, in only half of the fish LH cells responded. Each dot represents one fish (n=7, 4 males, 3 females, unpaired t-test, * p<0.05).

The stimulated calcium activity of LH and FSH cells is associated with hormone secretion.

(A and B) Top: Graphs showing mean calcium trace of 10 LH cells (left panel) or FSH cells (right panel) from consecutive imaging sessions before, during, and after the application of the stimulus (GnRH or CCK). Bottom: Secretion of LH or FSH before or after GnRH (A) or CCK (B) stimulation. Compared to basal levels, GnRH increased LH secretion, whereas CCK increased FSH secretion (dots from the same imaged pituitaries are connected with a line; n=5, paired t-test, *p<0.05). (C) Two hours after injection into live fish (n=10, 5 females and 5 males), CCK, but not GnRH, significantly increased the transcription of FSH in the pituitary (see Supplementary Fig. 4 for LH transcription; one-way ANOVA, * p<0.05, ** p<0.01). (D) FSH plasma levels increased significantly in a dose dependent manner after CCK injection (n=10, 5 females and 5 males), whereas GnRH injection did not affect FSH secretion (one-way ANOVA, * p<0.05, *** p<0.001).

A model summarizing the two suggested regulatory axes controlling fish reproduction.

The satiety-regulated CCK neurons activate FSH cells. LH cells are directly regulated by GnRH neurons that are gated by CCK, photoperiod, temperature, and behaviour, eventually leading to final maturation and ovulation. Bottom image schematically represents the relative timescale of the two processes and the associated gonadotropin levels. (Created with BioRender.com)

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