Knocking-down of the Prokineticin receptor 2 affects reveals its complex role in the regulation of the hypothalamus-pituitary-gonadal axis in the zebrafish model

Abstract

Prokineticin receptors (PROKR1 and PROKR2) are G protein-coupled receptors which control human central and peripheral reproductive processes. Importantly, allelic variants of PROKR2 in humans are associated with altered migration of GnRH neurons, resulting in congenital hypogonadotropic hypogonadism (CHH), a heterogeneous disease characterized by delayed/absent puberty and/or infertility. Although this association is established in humans, murine models failed to fully recapitulate the reproductive and olfactory phenotypes observed in patients harboring PROKR2 mutations. Here, taking advantage of zebrafish model we investigated the role of prokr1b (ortholog of human PROKR2) during early stages of GnRH neuronal migration. Real-Time PCR and whole mount in situ hybridization assays indicate that prokr1b spatial-temporal expression is consistent with gnrh3. Moreover, knockdown and knockout of prokr1b altered the correct development of GnRH3 fibers, a phenotype that is rescued by injection of prokr1b mRNA. These results suggest that prokr1b regulates the development of the GnRH3 system in zebrafish. Analysis of gonads development and mating experiments indicate that prokr1b is not required for fertility in zebrafish, although its loss determine changes also at the testis level. Altogether, our results support the thesis of a divergent evolution in the control of vertebrate reproduction and provide a useful in vivo model for deciphering the mechanisms underlying the effect of PROKR2 allelic variants on CHH.

Figure 1

Real-time-PCR experiments show that during GnRH3 neuron development prokr1a (A) is less expressed compared to prokr1b (B). (gene were normalized using eef1a as housekeeping gene. For each developmental stage n = 20).

Figure 2

Whole-mount in situ hybridization with prokr1a probe (A–F) did not display any signal in the head of zebrafish embryos. In contrast, prokr1b (G–L) is expressed in cells near the olfactory region at 36 hpf (G,H), 48 hpf (I,J) and 72 hpf (K,L). That is similar to gnrh3 expression at these times (M–R). Panels A,C,E,G,I,K,M,O and Q show lateral views. Panels B,D,F,H,J,L,N,P and R show dorsal views of zebrafish embryos. Scale bar = 50 µm. Image taken using Leica Application Suite software (LAS version 4.7.0; https://www.leica-microsystems.com/products/microscope-software/p/leica-application-suite/).

Figure 3

Injection of ctrl-MO (B) or prokr1a-MO (C) does not affect GnRH3 neuron fibers, as they look comparable to uninjected WT (A) embryos. In contrast, knockdown of prokr1b (D) causes alteration in axons migration of the rostral part (dotted square in Fig A) and at the level of the anterior commissure (AC) of the embryos. OB = olfactory bulbs, OC = optic chiasm, AC = anterior commissure. Scale bar=50 µm.

Figure 4

prokr1b homozygous mutant zebrafish (tg(gnrh3:EGFP);prokr1b^−/− (C,G) exhibit misrouting of rostral (dotted square) and optic chiasm (OC) fibers. These defects are not present in WT (A,E) and prokr1b heterozygous mutants (tg(gnrh3:EGFP);prokr1b^+/- (B,F). The prokr1b mutant phenotype is rescued by injection with WT prokr1b mRNA (D,H). OB = olfactory bulbs, OC = optic chiasm, AC = anterior commissure. Scale bar = 50 µm.

Figure 5

Quantification of GnRH3 fiber network confirms that prokr1b^−/− embryos have reduced GnRH3 neuron projections, and that this phenotype is rescued by injection of prokr1b mRNA when analyzed at 48 hpf (A) or 72 hpf (B). For each group n = 10 animals. ns: not significant, ***p < 0.01.

Figure 6

Histological sections conducted on 3 months of age fish showed no defects in the structure of gonads and testis between WT (A,C), heterozygous (E,G) and homozygous mutant (I,K). Sections zoomrevealed fully mature oocytes (black arrows in B,F,J) and fully mature spermatozoa (yellow arrows in D,H,L) in WT,prokr1b^+/− and prokr1b^−/− animals. Comparison of reproductive outputs (M) and GSI index (N) revealed no differences between WT, heterozygous and homozygous mutants. Real-Time PCR conducted on brain (O) and gonads (P) of WT and mutant fish showed a different expression of lhβ, fshβ, lhr and fshr in prokr1b^−/− males. Image taken using Leica Application Suite software (LAS version 4.7.0; URL: https://www.leica-microsystems.com/products/microscope-software/p/leica-application-suite/).