Loss of responses to odorants and pheromones in mPRγ (paqr5b)-knockout zebrafish

Abstract

In our previous study, we generated a membrane-type progesterone receptor γ (paqr5b) knockout zebrafish line. Knocking out paqr5b by genome editing resulted in the loss of neurons in the olfactory rosette (OR). These findings indicated that Paqr5b plays an essential role in the formation of olfactory neurons. In this study, we investigated the extent to which paqr5b^−/− fish lacking olfactory neurons retain their sense of smell. We used a reported tank for zebrafish olfactory analysis with a dividing plate in the middle. The tank was divided into three zones: the right zone, where test substances were added; the neutral zone; and the left zone. The fish were released into the neutral zone at the beginning of each test, the chemical was added to the corner of the right zone, and a 3-min video was taken to track the movements of the fish. The video was then played back, and the time spent in the three zones was counted manually. Both male and female paqr5b^+/+ and paqr5b^−/− zebrafish were separately analyzed for time spent in the three zones after exposure to ATP, cadaverine, and the pheromone 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Both male and female paqr5b^+/+ zebrafish were strongly attracted to ATP and stayed in the right zone for approximately 2 min (67%). In addition, paqr5b^+/+ zebrafish avoided cadaverine and stayed longer in the left zone than in any other zone. In contrast, paqr5b^−/− zebrafish stayed in all three zones for approximately the same amount of time, even after exposure to ATP and cadaverine. The paqr5b^+/+ fish were attracted to DHP and stayed in the right zone longer. Paqr5b^−/− zebrafish of either sex were not reactive to DHP exposure. These results showed that paqr5b^−/− zebrafish lacking olfactory neurons lost responsiveness to odorants and pheromones.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10695-025-01563-2.

Introduction

Zebrafish have become an important animal model for studying toxicity, disease, and human development. The discovery of thousands of early developmental mutations has made zebrafish a widely used model in developmental biology (Lieschke and Currie 2007). Membrane progesterone receptors (mPRs), which serve as receptors for hormones that trigger oocyte maturation, were first identified in the ovary (Tokumoto et al. 2004). Membrane progesterone receptors are also found in many different tissues and organs throughout the body, although they were first identified in the ovaries of fish. Consequently, mPRs are thought to contribute to a variety of rapid progesterone actions triggered from the cell surface through multiple intracellular signaling pathways. Previous research has shown that seven paqr genes affect the induction of oocyte maturation in paqr knockout zebrafish (Wu et al. 2020). The five mPRs (mPRγ, δ, α, β, and ε) are members of the progestin and adipoQ receptor (PAQR) family (paqr5 to 9). mPRδ (paqr6) and mPRε (paqr9) are abundantly expressed in the brain (Thomas and Pang 2012). Different mPRs are expressed in the brain and pituitary in the Chinese black sleeper (Bostrichthys sinensis), rainbow trout, European eel (Anguilla anguilla), and channel catfish (Ictalurus punctatus) (Mourot et al. 2006; Zhang et al. 2016). According to previous research from our laboratory, zebrafish exhibit remarkable morphological and functional changes in olfactory sensory neurons (OSNs) when mPRγ (paqr5b) is knocked out by genome editing (Mustary et al. 2024). Paqr5b^−/− zebrafish have shortened brain lengths, with the cerebellum positioned anteriorly and overlapping with the midbrain. In addition, these fish have underdeveloped olfactory rosettes (ORs) without neuronal cells. Micro-CT imaging revealed filamentous structures that connect the ORs to the olfactory bulb. Histological observations of ORs and fluorescent immunohistochemical analyses revealed a significant decrease in cell numbers in the lamellae of paqr5b^−/− zebrafish ORs. The OR of paqr5b mutant fish lacks crypts, microvilli, and ciliated olfactory sensory neurons (OSNs).

Animal survival depends heavily on the ability of the olfactory system to recognize and discriminate a wide variety of odor cues and to mediate critical actions such as feeding, mating, social behavior, and risk assessment (Firestein 2001; Kowatschew and Korsching 2021). The olfactory system must be highly dynamic to adapt odor-mediated actions rapidly to changing environments; therefore, it has many plasticity mechanisms that allow it to adapt continuously to odors (Whitlock 2006). In addition, olfactory organs have the ability to detect neurotoxic chemicals, heavy metals, contaminants, and infectious agents. This highlights the importance of olfactory plasticity (Tierney et al. 2010).

Here, we investigated the olfactory detection ability of paqr5b^−/− zebrafish. The aquatic environment provides enticing compounds that can guide fish to mates or food. The brain circuit and olfactory receptor that detect the scent of adenosine triphosphate have recently been identified. The OSN, which is highly sensitive to ATP and related chemicals, relays olfactory information to a part of the forebrain that is responsible for initiating appropriate foraging actions (Kowatschew and Korsching 2021; Wakisaka et al. 2017). Cadaverine, a diamine produced by the bacterial decarboxylation of lysine during putrefaction of dead fish, was found to be a potent stimulator of olfactory receptors in zebrafish (Hussain et al. 2013). Polyamines, such as cadaverine, are strong olfactory stimuli for goldfish, according to electroolfactogram (EOG) recordings (Rolen et al. 2003). In addition, we used DHP (17α,20β-dihydroxy-4-pregnen-3-one) to evaluate their pheromone response. Previous work in our laboratory has shown that DHP can induce sexual behavior in males and that females can respond by ovulating and producing eggs (Ahamed et al. 2024).

In this study, we investigated the olfactory ability of paqr5b^−/− zebrafish by using odorants and a pheromone. The results of this study demonstrated that paqr5b^−/− zebrafish lost their olfactory ability as a result of olfactory neuron loss.

Materials and methods

Materials

ATP was purchased from Yamasa Shoyu Co. (Chiba, Japan). Cadaverine was purchased from Sigma-Aldrich Chemicals (St. Louis, MO). DHP was purchased from Toronto Research Chemicals (Toronto, Canada).

Zebrafish

The original strains of roy (mpv17^a9/a9) and albino (golden-2; brs^b2/b2) were generously donated by Dr. K. Kawakami. A mutation in the paqr5b gene induced by genome editing in the roy strain was introduced into the albino strain by crossing. Albino siblings without mutation (paqr5b^+/+) and mutant (paqr5b^−/−) strains (Mustary et al. 2024) were used in this study. Zebrafish were cultivated following the standard protocol (Westerfield 1995). The zebrafish were housed in a tank with the water temperature set at 28.5 °C under a 14:10 light/dark cycle, and the zebrafish were fed brine shrimp in the morning and instant dry food, Tetra Guppy (Tetra GmbH, Melle Germany) in the evening. All the methods were carried out in accordance with relevant institutional guidelines and regulations.

Odorant-dependent behavior analysis using an experimental device

The tank with a divider in the middle that was used for the zebrafish olfactory analysis was custom-made (Hussain et al. 2013). The tank dimensions were as follows: height, 11.8 cm; width, 16.5 cm; and length, 29 cm (Fig. 1A). The internal partition was 20 cm long. The left arm, right arm, and neutral zone were the three separate sections of the experimental tank compartment separated by a central tank wall (20 cm). The fish were released into the neutral zone. At the beginning of each run, chemicals were applied to the corner of the right zone. To track fish movement, 3 min of video was recorded for each trial, and the time spent in all three areas (the left zone, right zone, and neutral zone) was manually counted (Godoy et al. 2020). ATP, cadaverine, and DHP were dissolved in ethanol at 10,000 times the stock concentration and used at final concentrations of 10 µM for ATP and cadaverine, while DHP was used at a concentration of 1 nM. The concentrations of ATP and cadaverine were set according to the published concentrations in the papers (Dieris et al. 2017; Wakisaka et al. 2017). The DHP concentration was set according to the published working concentrations in goldfish (Kobayashi et al. 2002). The working concentrations in zebrafish were determined after preliminary experiments.

Fig. 1.

Experimental device and fish. A Experimental device. The test tank dimensions were as follows: height, 11.8 cm; width, 16.5 cm; and length, 29 cm. The internal divider had a length of 20 cm. The three zones of the experimental tank, i.e., the left zone, right zone, and neutral zone, are indicated. The position where the compounds were applied is indicated in red. B Morphology of paqr5b fish. Representative image of the head region and dissected ORs of the wild-type (paqr5b^+/+) and mutant (paqr5b^−/−) zebrafish. Scale bars are 1 mm for the head region (white) and 200 µm for the OR (black)

Olfactory rosette dissection

Olfactory rosette dissections were carried out under a microscope with fine forceps in zebrafish Ringer’s solution (Mustary et al. 2024).

Results

Wild-type siblings (paqr5b^+/+) and paqr5b^−/− zebrafish with abnormal head morphology were used in this experiment (Fig. 1B). The paqr5b^−/− zebrafish had a very small OR with neuronal loss inside the depressed head (Fig. 1B). First, we tested the odorants ATP, which has been shown to attract zebrafish, and cadaverine, which has been shown to induce escape behavior in zebrafish (Fig. 2). We also examined the reactivity to DHP, a pheromone known to induce reproductive behavior in fish.

Fig. 2.

Behavioral analysis results of paqr5b after compound addition. The total time spent in three separate areas (left, neutral and right zones) after the addition of ATP (A), cadaverine (B), or DHP (C) is indicated. The results for the wild-type (paqr5b^+/+) and mutant (paqr5b^−/−) zebrafish are represented by open or closed bars, respectively. For each compound, 15 trials involving 5 fish of each sex were conducted. Each value represents the mean of the data, and the vertical lines indicate the standard deviation. The significant differences in the time spent in each zone between the wild type and the mutant zebrafish are indicated (**P ≤ 0.001, ***P ≤ 0.0001, ****P ≤ 0.00001)

Paqr5b^+/+ fish were strongly attracted to ATP. In contrast, paqr5b^−/− fish were not attracted to ATP (Fig. 2A) (Movies 1 and 2; movies are edited at 18 × speed). Paqr5b^+/+ fish of both sexes stayed in the right zone where ATP was added for approximately 2 min out of 3 min. In contrast, in paqr5b^−/− zebrafish, both sexes stayed in the three zones for approximately the same amount of time (approximately 1 min each). In addition, both groups stayed in the neutral zone for approximately the same amount of time. These results suggest that the olfactory response to ATP was lost in paqr5b^−/− zebrafish.

Fish exhibit repellent behavior toward cadaverine (1,5-pentanediamine), a component of an odor generated during fish decomposition. In this study, paqr5b^+/+ males and females exhibited strong avoidance behavior toward cadaverine, staying longer in the left zone than in the right zone, where cadaverine was added and rarely stayed in the right zone (Fig. 2B) (Movies 3 and 4; movies are edited at 18 × speed). In this case, females showed stronger avoidance behavior. However, paqr5b^−/− fish did not react to cadaverine and did not show significant differences in time spent among the three zones.

Additionally, the paqr5b^+/+ fish responded to DHP, a hormone that induces oocyte maturation in fish and is also known to induce reproductive behavior. Paqr5b^+/+ males and females stayed longer in the right zone, to which DHP was added. Paqr5b^−/− fish were not responsive to DHP and remained in all three zones for approximately the same amount of time (Fig. 2C) (Movies 5 and 6; movies are edited at 18 × speed).

Discussion

Our previous study revealed that the OR in paqr5b^−/− zebrafish is undeveloped and small in size (Mustary et al. 2024). Consequently, paqr5b gene knockout fish have no olfactory sensory neurons in their ORs. These findings suggest that their ability to detect odors and pheromones is severely impaired or absent. Therefore, we analyzed the extent to which paqr5b^−/− fish are able to detect the odors and pheromones necessary for mating, tracking, and foraging.

Fish generally exhibit positive chemotaxis to nucleotides that induce feeding behavior, particularly to ATP dissolved in food. A previous study reported that the receptor that accepts ATP as a cue is the adenosine receptor A2c, which is coexpressed with c-Fos, a neuronal activation marker, in pear-shaped OSNs (Wakisaka et al. 2017). ATP is actually degraded to adenosine by tissue-nonspecific alkaline phosphatase (TNAP; alpl) and ecto-5-nucleotidase (CD73; nt5e) on the OR surface before binding to A2c, but stimulation by ATP in the water also increases c-Fos expression in neurons in the brain. The loss of neurons, including pear-shaped OSNs, in paqr5b^−/− zebrafish resulted in a loss of responsiveness to ATP.

In zebrafish, TAAR13c, one of the trace amine-associated receptors (TAARs), is a receptor for cadaverine, a component of the odor generated during fish decomposition that is a danger signal for fish (Dieris et al. 2017). TAAR13c was found to be expressed in only a small percentage of the neurons in the OR. These findings indicate that TAAR13c is involved in the response to a specific group of odorants, with localized receptor expression among similarly shaped ciliated neurons. In paqr5b^−/− fish, almost all ciliated neurons were lost, resulting in their nonresponsiveness to cadaverine. The receptor for DHP has not yet been identified. Recently, we synthesized the zebrafish Paqr5b protein and investigated its hormone-binding properties (Sohan et al. 2025). Our results showed that Paqr5b binds extensively to progesterone and other known neurosteroids, such as allopregnanolone (ALLO). Paqr5b was expressed in almost all neurons in the normal ORs of wild-type zebrafish (Mustary et al. 2024). The present study revealed that paqr5b^−/− neurons are defective, leading to the loss of responsiveness to DHP. These results suggest that Paqr5b functions as a receptor for progesterone pheromones such as DHP. Previously, a receptor for prostaglandin F_2α, OR114-1, was identified and shown to be expressed in ciliated olfactory sensory neurons (Yabuki et al. 2016). A similar analysis should be conducted for Paqr5b.

The differentiation mechanism of olfactory sensory neurons (OSNs) was initially studied in mammals and has since been examined in fish. Recent analyses of gene-knockout zebrafish have revealed that the transcription factor Foxj1 is essential for cilia biogenesis in olfactory cells. Foxj1 mutants exhibit abnormal cilia biogenesis and diminished bile acid responses (Rayamajhi et al. 2024). IFT88 mutants, which are members of the intraflagellar transport (IFT) gene family, exhibit abnormal olfactory cell cilia formation. Cilia defects are followed by sensory cell degeneration (Bergboer et al. 2018). Studies have shown that the neural crest-specific gene Sox10 is necessary for microvillous neurogenesis when cells ingress into the olfactory epithelium (Cheung et al. 2021; Saxena et al. 2013). Previous studies have shown that paqr5b knockout fish lose all OSNs, a finding supported by the absence of a smell phenotype observed in this study. It is expected that the Paqr5b-mediated signal is involved in the early steps of differentiation. Further investigation is necessary to determine when OSN loss occurs in the Paqr5b knockout developmental process.

Zebrafish have only 60 olfactory receptor genes, whereas the human genome has approximately 1000, making it the largest superfamily among mammals (Buck and Axel 1991; Bushdid et al. 2014). Researchers are now studying the human olfactory system to predict Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Bowman 2017; Lafaille-Magnan et al. 2017; Liao et al. 2024; Morley and Duda 2010; Ubeda-Bañon et al. 2020). Apparent changes have been observed in the olfactory regions of patients’ brains with PD or AD. Thus, researchers are seeking a way to detect PD or AD during the preclinical stage. Another area of research on the human olfactory system involves the ectopic expression of olfactory receptors in various tissues and their relationship to diseases in those tissues (Shi et al. 2024). The cardiovascular system is the most extensively studied, with olfactory receptors detected in each tissue and cell and signal transduction pathways from various ligands analyzed. These disorders are thought to be related to cardiovascular disease.

We found that paqr5b^−/− fish showed no responsiveness to the odorants ATP and cadaverine and the pheromone DHP. We generated knockout fish lines for all seven paqr genes in zebrafish and found no significant OR abnormalities in any of the lines except paqr5b. The seven paqr genes are widely expressed throughout the body, and in many tissues, the knockout of a single gene may be compensated for by mutual redundancy. However, paqr5b was abundantly expressed in the OR; this may have resulted in a noticeable phenotype in the OR (Saraiva et al. 2015).

The lack of responsiveness to odorants was expected since odorants are sensed by olfactory neurons in ORs. Our immunostaining analysis revealed that Paqr5b is expressed on the cell surface of the three major olfactory neurons (Mustary et al. 2024). Additionally, we have demonstrated that Paqr5b binds to progesterone as well as to the neurosteroid, allopregnanolone (ALLO) in experiments involving recombinant Paqr5b proteins (Sohan et al. 2025). It is likely that Paqr5b acts as a receptor for these compounds. However, neural region or cell activation by DHP or ALLN treatment must be detected to confirm Paqr5b’s role as a receptor in olfactory cells, as demonstrated with TAAR, the cadaverine receptor (Dieris et al. 2017). However, since OSNs are completely lost in the paqr5b mutant, it is unclear if Paqr5b functions as a DHP receptor.

Conclusion

In this study, we performed a behavioral analysis to evaluate the olfactory ability of paqr5b^−/− zebrafish using several odorants and a pheromone. The paqr5b^−/− zebrafish failed to respond to odorants and pheromones. Neurons were lost in the OR of paqr5b^−/− zebrafish. The results revealed that paqr5b^−/− zebrafish lost their sense of smell due to the loss of neurons in the OR.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ATP

Adenosine triphosphate

DHP

17α,20β-Dihydroxy-4-pregnen-3-one

EOG

Electroolfactogram

GPCR

G protein-coupled receptor

mPR

Membrane progesterone receptor

OR

Olfactory rosette

OSN

Olfactory sensory neuron

PAQR

Progestin and adipo-Q receptor

Author contribution

MEH maintained the zebrafish strains, performed the behavior analysis, analyzed the data and drafted the manuscript. SA performed the behavior analysis. MMH edited the videos. MTA summarized the data. UHM established the mutant strain. TT participated in the study design, supervised the study and wrote the paper. All the authors read and approved the final manuscript.

Funding

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan and JSPS KAKENHI Grant Number 23K05830 (to TT). This work was supported by NIG-JOINT (29A2024) to TT. This work was also supported by JST, the establishment of university fellowships toward the creation of science technology innovation, Grant Number JPMJFSFS119. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We also received student scholarship from the Japanese Government (MEXT) Scholarship for MT Amin; Bangabandhu Science and Technology Fellowship Trust, Ministry of Science and Technology, Government of the People’s Republic of Bangladesh for S Ahamed (memo no. 08//04–01-2021).

Data availability

No datasets were generated or analysed during the current study.

Code availability

Not applicable.

Declarations

Ethical approval

All zebrafish experiments were carried out with approval from the Institutional Ethics Committee of Shizuoka University, Japan (approval no. 2023F-9 and 2024F-9); the guidelines set by this committee for the use of animals were strictly followed.

Consent for publication

All the authors reviewed and approved the manuscript for publication.

Competing interests

The authors declare no competing interests.