Prokineticin receptor 2 affects gnrh3 neuron ontogeny but not fertility in zebrafish

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. SIMILAR CONTENT BEING VIEWED BY OTHERS DISRUPTING AMH AND ANDROGEN SIGNALING REVEALS THEIR DISTINCT ROLES IN ZEBRAFISH

GONADAL DIFFERENTIATION AND GAMETOGENESIS Article Open access 05 March 2025 LOSS-OF-FUNCTION VARIANTS IN _SEMA3F_ AND _PLXNA3_ ENCODING SEMAPHORIN-3F AND ITS RECEPTOR PLEXIN-A3 RESPECTIVELY

CAUSE IDIOPATHIC HYPOGONADOTROPIC HYPOGONADISM Article 25 January 2021 COMMON AND FEMALE-SPECIFIC ROLES OF PROTEIN TYROSINE PHOSPHATASE RECEPTORS N AND N2 IN MICE REPRODUCTION Article Open

access 07 January 2023 INTRODUCTION The hypothalamus-pituitary-gonadal (HPG) axis controls reproduction in vertebrates1,2,3,4,5 through the pulsatile release of gonadotropin-releasing

hormone (GnRH) hormone by GnRH neurons. During development, these neurons differentiate from neural crest cells and ectodermal progenitors in a niche at the border between the respiratory

epithelium and the vomeronasal/olfactory epithelium. From these regions these neurons migrate caudally to reach the medio-basal hypothalamus, where they complete their differentiation6.

Several studies in different animal models have shown that specific evolutionarily conserved genes regulate this migratory process and the functions of these neurons. The role of these genes

in human fertility is supported by the existence of variants in these genes associated with congenital hypogonadotropic hypogonadism (CHH)7, a rare and clinically heterogeneous disorder

characterized by abnormal pubertal development and/or infertility, a normal (nCHH) or defective sense of smell (Kallmann syndrome, KS) and other reproductive and non-reproductive

anomaliess8,9,10. More than 25 genes have been associated with CHH, although variants in these genes account only for 40–50% of reported cases. Several evidences indicate that CHH is a

complex genetic disease characterized by variable expressivity and penetrance of the associated genetic defects, which can be partially explained by an oligogenicinheritance model9,11. Among

the genes involved in CHH, prokineticin receptor 2 (_PROKR2)_ has an important role in GnRH neuron migration. PROKR2, as a member of the GPCR family, has an extracellular amino-terminal

end, an intracellular carboxy-terminal domain and a central core formed by seven transmembrane α-helical segments (TM1–TM7)12. Prokineticins have been previously demonstrated to be involved

in several physiological functions in neurogenesis, regulation of circadian rhythms, metabolism, angiogenesis, pain perception, muscle contractility, hematopoiesis, immune response,

thermoregulation and energy expenditure13,14,15. Matsumoto and colleagues in 2006 reported the first observation that _Prokr2_ knock-out mice display hypoplastic gonads and olfactory

structures, a phenotype reminiscent of KS, to link this gene to CHH16. Furthermore, _Prokr2_ knock-out mice show decreased plasma levels of testosterone and follicle-stimulating hormone, but

not luteinizing hormone. In humans, genetic screening of CHH cohorts has revealed mutations in _PROKR2_ in KS and nCHH patients, but mostly in the heterozygous state17,18,19,20. Moreover,

studies on transfected cells demonstrated that the missense _PROKR2_ variants observed in KS and nCHH patients have a deleterious effect on _PROKR2_ signaling18,20,21 although such variants

are also present in apparently unaffected individuals22,23,24. Thus, despite several _in vitro_ and _in vivo_ studies, the role of Prokr2 in GnRH neuron function and in CHH pathogenesis

remains incompletely understood. Here, we take advantage of the zebrafish to generate an _in vivo_ model to investigate _PROKR2_ function during GnRH neuron ontogeny. Using both transient

knockdown and germline knockout of _prokr1b_, the zebrafish ortholog of human _PROKR2_, we find that _prokr1b_ has an important role in the migration of GnRH axons, but not for fertility, in

this animal model. RESULTS Mammals possess two prokineticin receptors named _PROKR1_ and _PROKR2_25. The zebrafish genome contains two _prokr_ paralogs named _prokr1a_ and _prokr1b_ (or

_prokr1l_), and previous evidence suggests that _prokr1a_ and _prokr1b_ correspond to mammalian _PROKR1_ and _PROKR2_, respectively26. To determine which of these genes may be involved in

GnRH neuronal migration, we evaluated their expression during zebrafish embryo development. Real-Time PCR analysis on RNA extracts from whole embryos revealed that _prokr1b_ is expressed at

higher levels compared to _prokr1a_ and exhibits an increase from 24 hours post-fertilization (hpf) to 72 hpf (Fig. 1A,B). Whole-mount _in situ_ hybridization (WISH) performed at the same

developmental stages (Fig. 2) revealed that _prokr1b_, but not _prokr1a_, is expressed in cells adjacent to the olfactory placodes (Fig. 2G-L), similar to the pattern observed for _gnrh3_

(Fig. 2M-R). Given the role of _PROKR2_ in the migration of GnRH neurons in humans, these results suggest that _prokr1b_ may similarly be involved in GnRH3 neuron development in zebrafish.

Next we used morpholino anti-sense oligonucleotides (MOs) to knock-down expression of _prokr1a_ or _prokr1b_ in _tg (gnrh3:EGFP)_ embryos, which express EGFP in GnRH3 cells27 (Supplementary

Fig. S1A–C). Images collected at 48 hpf (Fig. 3) revealed that _prokr1a_ knock-down (Fig. 3C) did not affect the development of GnRH3 fibers that appeared similar to those of uninjected

wild-type animals (WT, Fig. 3A) or injected with a control MO(_ctrl-MO)_ (Fig. 3B). In contrast, knock-down of _prokr1b_ led to evident alterations in the architecture of the GnRH3 network

(Fig. 3D). In these embryos, GnRH3 fibers appeared disorganized, especially at the level of the anterior commissure (AC) and in the anterior fibers (dotted square in Fig. 3D; Supplementary

Fig. S1D). These results suggest that _prokr1b_ is required for normal GnRH neuron development in zebrafish. To confirm the defects observed for _prokr1b_ knock-down using MOs, a technique

that is prone to non-specific artefacts, we next analyzed GnRH3 neuron development in animals containing a germline mutation in _prokr1b_26. To do so, we compared EGFP-expressing GnRH3

neuron fibers in _prokr1b_ homozygous mutant, heterozygous mutant, and homozygous WT siblings at 48 hpf and 72 hpf. No differences were observed between _prokr1b_+_/−_ and WT siblings at

these two time points (Fig. 4A,B,E,F), while _prokr1b_−/− embryos (Fig. 4C,G) showed defects in GnRH3 neuron fibers in the same anatomical region as in the knockdown experiments (Fig. 3D).

This phenotype appears to be specific to the absence of _prokr1b_, as injection of WT _prokr1b_, but not _prokr1a_ mRNA into _prokr1b_−/− animals at the 1-cell stage rescued the phenotype

(Fig. 4D–H; Supplementary Fig. S2E,F). These observations were supported by quantitative analysis of GnRH3 neuron fibers(Fig. 5A,B), providing an evidence for a role of _prokr1b_ in GnRH

neuron development in zebrafish. In order to establish whether _prokr1b_ is required for the development and function of the reproductive system in zebrafish, as it is in humans and mice, we

compared the reproductive organs and fecundity of _prokr1b_−/−, _prokr1_+_/_- and _prokr1b_+/+ siblings. Histological sections of testes and ovaries of 3-months-old male and female animals

did not reveal obvious differences among the three genotypes, suggesting that _prokr1b_ is not required for gonadal maturation in both sexes and, by consequence, for puberty in zebrafish

(Fig. 6A-L). No differences were also observed among the three genotypes in the number of fertile eggs generated (Fig. 6M), neither in the GSI index of mutated male or female compared to WT

indicating that _prokr1b_ is not fundamental for fertility in zebrafish (Fig. 6N). Nevertheless, Real-Time qPCR conducted on tissue of adult fish, revealed for mutated males higher

expression of _lhβ_ and _fshβ_ in the brain (Fig. 6O) together with a strong decrease of the gonadotropic receptors _lhr_ and _fshr_ in the gonads (Fig. 6P). DISCUSSION Several recent

studies have demonstrated a remarkable evolutionary conservation of the developmental migration of GnRH neurons and of several genes involved in GnRH ontogeny28,29,30,31. In zebrafish, like

in mammals, GnRH secreting neurons starts their development at 24 hpf from cells located in the olfactory epithelium that send dorsal extensions that ultimately innervate the hypothalamus

and pituitary. An important gene involved in the development of the GnRH system in humans is _PROKR2_. The zebrafish genome contains two _prokineticin receptor_ paralogues, _prokr1a_ and

_prokr1b_26. WISH analyses reveal that _prokr1b_ expressionstarts in the brain at 24 hpf close to the olfactory bulbs and appears similar to that of _gnrh3_ at 48 and 72 hpf. Accordingly,

Real-Time PCR data show that during this time window _prokr1b_ expression increases and then drastically decreases at 96 hpf. At 72 hpf, the development of GnRH3 fibers is complete and is

followed by the migration of GnRH3 somata from the olfactory region to the hypothalamus27. Consistent with the possibility that zebrafish _prokr1b_ is an ortholog of human _PROKR2_,

knockdown of _prokr1b_, but not _prokr1a_, affected the formation of rostral GnRH3 fibers, similar to humans with _PROKR2_ mutations. Importantly, similar defects were also present in

homozygous _prokr1b_ mutant embryos at 48 hpf, and this phenotype was rescued by injection of WT _prokr1b_ mRNA. Taken together, these results demonstrate that _prokr1b_ is important for the

correct migration of GnRH3 neuron fibers. Although _prokr1b_ appears to be the zebrafish ortholog of human and murine _PROKR2_, the zebrafish _prokr1b_ mutant phenotype does not fully

recapitulate the clinical features of CHH. Indeed, _prokr1b_ mutation does not affect gonadal maturation or fertility in zebrafish, as demonstrated by fecundity testing and histological

analysis of testis and ovaries at 3 months of age. Moreover, dorsoventral projections of GnRH3 neurons, despite reduced, are present in zebrafish _prokr1b_ mutants at 72 hpf, in contrast to

murine _Prokr2_ mutants in which there is an early arrest in GnRH neuronal migration16. Two recent studies conducted in zebrafish have highlighted differences in the role of the GnRH system

during puberty and fertility. Liu and colleagues showed that triple mutants lacking _gnrh3_ and the 2 kisspeptin ligands undergo normal puberty and gonad maturation32. These results are

surprising, because GnRH3 has been considered the most important stimulator of gonadotropin release in fish and its expression, together with _kiss1_ and _kiss2_, have been found to be

higher during puberty and gonadal maturation in zebrafish33. Moreover Marvel and colleagues demonstrated that zebrafish _gnrh2_/_gnrh3_ double mutants show normal fertility, demonstrating

that neither GnRH2, nor Kiss1 and Kiss2, compensate for loss of GnRH3 in zebrafish34. Nevertheless, comparison between WT and double or triple mutants revealed in both studies different

expression patterns of neuropeptides known to be important in mammal control of reproduction, such as _tachykinin 3_, _secretogranin II_ and _neuropeptide Y_. These results suggest that, in

contrast with mammals, multiple factors act in parallel with GnRH to stimulate the reproductive axis in zebrafish32,34,35. Our results in the knockout male fish might further confirm this

hypothesis. Indeed, we reported a _lhr_ and _fshr_ lower expression in the testis that could firstly indicate a role of _prokr1b_ in this organ similar to what already observed in mice,

where absence of _Prokr2_ lead to a variable degree of compromised vasculature, even in the absence of evident structural gonadal modification36. Moreover, this could also be related to

primary testes defect that has been described, in association to those in the hypothalamus and pituitary, also in human male with CHH (Sykiotis _et al_. JCEM 2010). Secondly, this reduced

receptor expression might lead to a relative resistance to Lh and Fsh action which, in turn, might activates, through the negative feedback mechanism, the central compartment of the HPG

axis. Higher _lhβ_ and _fshβ_ expression levels in our male knockout fish, seem to confirm this stimulation, nevertheless they are not consequence of level modification in _gnrh3_ expression

levels. Thus, other factors from GnRH3 system might be implicated in the stimulation of the pituitary as previously suggested32,34,35. In conclusion, even if mechanisms controlling the HPG

and, by consequence, fertility have slightly diverged along evolution, these studies together demonstrate that genes regulating GnRH ontogeny present a certain degree of conservation among

humans, mice and zebrafish37,38. Indeed, despite the variable phenotypic features, the _Tg(gnrh3:EGFP);prokr1b__ct814/ct814_ line presented here suggests that _prokr1b_ is the orthologue of

human _PROKR2_, and demonstrates that its loss affects the development of GnRH neuronal fibers in zebrafish, asin humans, but also the expression of the _lhr_ and _fshr_ at the testes level,

thus indicating a complex implication of the prokineticin pathway in the HPG functionality. Moreover, this mutant lineis a useful _in vivo_ tool that, combined with mutant lines for other

GnRH related genes, could contribute to our understanding of the development of the GnRH system and the complex mechanisms underlying CHH and related diseases. METHODS ZEBRAFISH LINES AND

MAINTENANCE Zebrafish (_Daniorerio_) embryos obtained from natural spawning were raised and maintained according to established techniques39. All experiments with live animals were performed

at the University of Milan. All experimental protocols and methods were carried out in accordance with relevant guidelines and regulations of Good Animal Practice approved by the

institutional and licensing committee IACUC (Institutional Animal Care and Use Committee) and University of Milan by the Italian Decree of March 4th, 2014, n.26. Embryos were staged

according to morphological criteria40. Beginning from 24 hpf, embryos were cultured in fish water containing 0.003% PTU (1-phenyl-2-thiourea; Sigma–Aldrich, Saint Louis, MO) to prevent

pigmentation and 0.01% methylene blue to prevent fungal growth39. Wild-type (WT) zebrafish of the AB strain were obtained from the Wilson lab (University College London, London, United

Kingdom). The _tg (gnrh3:EGFP)_27 and _prokr1b_ct814/ct814 26 zebrafish lines have been previously described. REAL-TIME PCR Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was

performed on total RNA prepared from 20 zebrafish oocytes and embryos for each different developmental stages using the Total RNA Isolation Kit (Ambion, Thermo Fisher, Waltham MA) or the

RNAgents Total RNA Isolation System (Promega, Madison, WI), treated with DNase I RNase free (Roche, Basel, Switzerland) to avoid possible contamination from genomic DNA. RNA concentrations

and quality were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, USA). Total RNA (1 ug) was reverse transcribed to produce cDNA using

Superscript III reverse transcriptase (Invitrogen) primed with random hexamers, as described previously41. In all cases, a reverse transcriptase negative control was used to test for genomic

DNA contamination. The primers used for quantitative Real-Time PCRare listed in the Supplementary Table S1. _IN SITU_ HYBRIDIZATION Whole-mount _in situ_ hybridization (WISH) was performed

as described42. PCR products were cloned into the pGEM-T Easy vector (Promega, Table S2). The cDNA-containing plasmids were linearized and transcribed with T7 and SP6 RNA polymerase (Roche)

for antisense and sense riboprobe synthesis. Images of stained embryos were taken with a Leica MZFLIII epifluorescence stereomicroscope equipped with a DFC 480-R2 digital camera. KNOCKDOWN

EXPERIMENTS We tested one antisense morpholino oligonucleotide (MO) each for _prork1a_ and _prokr1b_ (Supplementary Table S3). Both were splice-blocking MOs synthesised by Gene Tools LLC

(Oregon, USA). Morpholinos were dissolved in Danieau’s solution (58 mMNaCl; 0,7 mMKCl; 0,4 mM MgSO4. H2O; 0,6 mMCa(NO3)2; 5 mMHepes pH 7.2) at 2 mM and stored at −80 °C. Embryos were

microinjected at the 1–4 cell stage with rhodamine dextran (Molecular Probes) co-injected as a tracer. As a control for non-specific effects, a standard control morpholino (_ctrl-_MO) was

injected, which targets the human _β-globin_ gene. Morpholinos were tested for efficacy and toxicity by injecting different doses in _tg(gnrh3:EGFP)_ embryos and evaluating them for

morphological defects (Supplementary Fig. S1A-C). After injection, embryos were raised in fish water at 28 °C and observed until the developmental stage of interest. Embryos that were to be

imaged after 24 hpf were treated with PTU. For imaging, embryos were anaesthetized using tricaine (ethyl 3-aminobenzoate methanesulfonate salt, Sigma; 25x stock = 0.08 g in 20 ml of

distilled H2O) in fish water. Injected embryos (morphants) were embedded at 48 hpf in UltraPure Low Melting Point Agarose (Thermo Fisher Scientific) and photographed using a confocal laser

scanning microscope (Nikon C2) with a 20x objective. GENERATION OF _TG(GNRH3:EGFP)_; _PROKR1B_ CT814/CT814 LINE AND RESCUE EXPERIMENTS We crossed _tg(gnrh3:EGFP)_27 animals to

_prokr1b_ct814/ct814 animals26 to generate the _tg(gnrh3:EGFP)_; _prokr1b_ct814/ct814 line. Fin clipping was performed to isolate genetic material from individual fish for genotyping

accordingly to what previous published in Chen and colleague26 (Table S4). The _prokr1b_ct814/ct814 fish contain a 1 bp deletion (nucleotide 12 of the open reading frame: 5’-C-3’), which

results in a change in reading frame after amino acid 4 and a premature stop codon after amino acid 13 compared to 396 amino acids for the wild-type (WT) protein. Rescue experiments were

performed by injecting 400 pg _prokr1b_ mRNA diluted in the Danieau’s solution into 1-cell stage embryos. LIVE-IMAGING OF GNRH3 FIBERS IN _PROKR1B_ KO EMBRYOS To assess the role of _prokr1b_

during GnRH3 fiber development, _prokr1b_ KO animals were embedded at 48 or 72 hpf in UltraPure Low Melting Point Agarose (Thermo Fisher Scientific) and analysed using a confocal laser

scanning microscope (Nikon C2+) with a 20x objective. GnRH3 fiber structure was assessed and 3D reconstructed using Fiji43. Due to the complexity of GnRH3 fibers, a specific region of

interest (ROI) was selected and analyzed at each developmental stage (Supplementary Fig. S2), with background fluorescence subtracted from each image (Supplementary Fig. S2B–D). The number

of green pixels within each ROI was used as a proxy for the amount of GnRH3 fibers. GONADS HISTOLOGY, FECUNDITY/FERTILIZATION RATESAND GSI For histological analysis, gonads from 3-months-old

fish were fixed in 4% paraformaldehyde (PFA), dehydrated, wax-embedded, cut into 8 µm sections using a microtome (Leitz 1516), and stained with eosin. Samples were imaged using a Leica

DM6000 B microscope equipped with a Leica 480 digital camera using the Leica Application Suite (LAS version 4.7.0). The assessment of fecundity (number of eggs released) and fertilization

rate (fraction of eggs that developed into an embryo), WT and mutant females and males at 3 months-old were paired in a spawning tray. After one hour, eggs were collected in 30% Danieau’s

solution and counted. The number of fertilized and unfertilized eggs was discerned using a dissecting microscope at 6 hpf. Twelve-months-old male and female fish were then dissected to

collect ovaries and testicles for gonadosomatic index (GSI) measurement and Real-time PCR. The GSI was calculated according to the formula (organosomatic index = organ weight × 100/body

weight)44. STATISTICAL ANALYSIS Statistical analyses in Fig. 5 was performed using one-way ANOVA with Dunnett’s post-hoc test using GraphPad PRISM version 6.0 (GraphPad, San Diego, CA). In

the graphs, *_P_ < 0.05, **_P_ < 0.01, ***_P_ < 0.001. CHANGE HISTORY * _ 15 MAY 2020 An amendment to this paper has been published and can be accessed via a link at the top of the

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index of adult zebrafish (Danio Rerio). _Zebrafish_ https://doi.org/10.1089/zeb.2013.0891 (2013). Download references ACKNOWLEDGEMENTS The study was supported by funds from IRCCS Istituto

Auxologico Italiano (Ricerca Corrente funds: 05C623_2016) and funds from University of Milan – Dept. of Clinical Sciences and Community Health (Piano di sostegno alla ricerca - Linea 2

Azione B). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * IRCCS Istituto Auxologico Italiano, Division of Endocrine and Metabolic Diseases & Lab. of Endocrine and Metabolic Research,

Milan, Italy Ivan Bassi, Francesca Luzzani, Federica Marelli, Valeria Vezzoli, Ludovica Cotellessa & Luca Persani * Department of Clinical Sciences and Community Health, University of

Milan, Milan, Italy Federica Marelli, Ludovica Cotellessa, Luca Persani & Marco Bonomi * Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,

USA David A. Prober * Department of Neurobiology, The George S. Wise Faculty of Life Sciences and Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv, Israel Yoav Gothilf Authors *

Ivan Bassi View author publications You can also search for this author inPubMed Google Scholar * Francesca Luzzani View author publications You can also search for this author inPubMed 

Google Scholar * Federica Marelli View author publications You can also search for this author inPubMed Google Scholar * Valeria Vezzoli View author publications You can also search for this

author inPubMed Google Scholar * Ludovica Cotellessa View author publications You can also search for this author inPubMed Google Scholar * David A. Prober View author publications You can

also search for this author inPubMed Google Scholar * Luca Persani View author publications You can also search for this author inPubMed Google Scholar * Yoav Gothilf View author

publications You can also search for this author inPubMed Google Scholar * Marco Bonomi View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

I.B. designed, performed and interpreted the experiments and wrote the manuscript. F.L., F.M., V.V. and L.C. performed and assisted in the experiments. D.P. provided the _prokr1b_ct814/ct814

mutant line and interpreted the experiments. M.B., Y.G., and L.P. conceived the study, supervised the publication, interpreted the experiments and wrote the manuscript. CORRESPONDING AUTHOR

Correspondence to Marco Bonomi. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bassi, I., Luzzani, F., Marelli, F. _et al._ Prokineticin receptor 2 affects GnRH3

neuron ontogeny but not fertility in zebrafish. _Sci Rep_ 10, 7632 (2020). https://doi.org/10.1038/s41598-020-64077-2 Download citation * Received: 19 October 2019 * Accepted: 07 April 2020

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