Sexually dimorphic distribution of kiss1 and kiss2 in the brain of yellowtail clownfish, Amphiprion clarkii

in Endocrine Connections
Authors:
Yan-yu Zhang Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Xian Zhang Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Shao-yang Bu Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Wei-wei Zhang Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Tian-xiu Li Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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De-cai Zheng Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Ze-xiang Huang Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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Qian Wang Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou, Hainan, China

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https://orcid.org/0000-0002-7062-8763

Correspondence should be addressed to Q Wang: 992856@hainanu.edu.cn

*(Y Zhang and X Zhang contributed equally to this work)

DOI:
https://doi.org/10.1530/EC-22-0136
Volume/Issue:
Volume 11: Issue 8
Article ID:
e220136
Article Type:
Research Article
Online Publication Date:
25 Jul 2022
Copyright:
© The authors 2022
Collections:
Reproduction
Open access

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Kisspeptin system was shown to be a key factor in mediating social stress and reproduction. Yellowtail clownfish, Amphiprion clarkii, is a hermaphrodite fish, whose sex determination and gonadal development are affected by the social status of individuals. The yellowtail clownfish is a fantastic animal model to explore sex determination, but the social status and precise distribution of kiss mRNAs in the brain of this species are unknown. Hererin, a novel in situ hybridization technique, RNAscope, was used to investigate the distribution of kiss1 and kiss2 expressions in the brain of yellowtail clownfish. The coronal planes of brain showed that the kiss1 signal was mainly present in dorsal habenular nucleus (NHd) and kiss2 mRNA was widely expressed in telencephalon, midbrain, and hypothalamus, especially in dorsal part of the nucleus of the lateral recess (NRLd). Additionally, kiss1 and kiss2 signals have sexually dimorphic distribution. The kiss1 mRNA was distributed in NHd, the telencephalon, and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of females but in NHd and NDLIl of males. kiss2 signals were stronger in females than that in males. The distribution of kiss1 and kiss2 neurons in NHd of habenula and NRLd of hypothalamus may suggest that kiss genes associate environmental signaling and reproductive function in yellowtail clownfish.

Abstract

Kisspeptin system was shown to be a key factor in mediating social stress and reproduction. Yellowtail clownfish, Amphiprion clarkii, is a hermaphrodite fish, whose sex determination and gonadal development are affected by the social status of individuals. The yellowtail clownfish is a fantastic animal model to explore sex determination, but the social status and precise distribution of kiss mRNAs in the brain of this species are unknown. Hererin, a novel in situ hybridization technique, RNAscope, was used to investigate the distribution of kiss1 and kiss2 expressions in the brain of yellowtail clownfish. The coronal planes of brain showed that the kiss1 signal was mainly present in dorsal habenular nucleus (NHd) and kiss2 mRNA was widely expressed in telencephalon, midbrain, and hypothalamus, especially in dorsal part of the nucleus of the lateral recess (NRLd). Additionally, kiss1 and kiss2 signals have sexually dimorphic distribution. The kiss1 mRNA was distributed in NHd, the telencephalon, and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of females but in NHd and NDLIl of males. kiss2 signals were stronger in females than that in males. The distribution of kiss1 and kiss2 neurons in NHd of habenula and NRLd of hypothalamus may suggest that kiss genes associate environmental signaling and reproductive function in yellowtail clownfish.

Keywords: kisspeptin; social stress; sexually dimorphic distribution; yellowtail clownfish; RNAscope

Introduction

Kisspeptin is an upstream regulator of the reproductive axis (hypothalamic–pituitary–gonadal, HPG axis) (1). Kisspeptin interacts with its receptor, KissR (G protein-coupled receptor 54, GPR54), resulting in the release of the gonadotropin-releasing hormone (GnRH) and further regulating the gonadotropic hormone (GtHs, including luteinizing hormone and follicle-stimulating hormone) secretion (2). The GtHs act on the gonads and affect sexual differentiation and gonadal development in teleosts (3). Furthermore, kisspeptin system is also involved in modulating certain cancers and vascular dynamics (4).

Kisspeptin is encoded by one gene (KISS1/Kiss1) in mammals, whereas two paralogous genes, kiss1 and kiss2, have been identified in almost all teleosts, including Nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), medaka (Oryzias latipes), chub mackerel (Scomber japonicus), rohu (Labeo rohita), Siberian sturgeon (Acipenser baerii), sapphire devil (Chrysiptera cyanea), rare minnow (Gobiocypris rarus), pejerrey (Odontesthes bonariensis), sea bass (Dicentrarchus labrax), orange-spotted grouper (Epinephelus coioides), and goldfish (Carassius auratus) (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Additionally, only one kisspeptin-encoding gene was identified in several pleuronectiforms (17). Utilizing RNA sequencing and genomics technology, the multiple gene encoding kisspeptin will be identified in more teleosts (18, 19). Different expression patterns of kiss1 and kiss2 indicate the distinct physiological functions they would play. In chub mackerel, kiss1 is mainly expressed in the brain, whereas kiss2 is expressed in the brain, pituitary, and testis (20). In yellowtail clownfish (Amphiprion clarkii), the highest kiss1 expression level is detected in the liver, but kiss2 is mainly expressed in the cerebellum, pituitary, and hypothalamus (21). In rare minnow with estradiol treatment, both kiss1 and kiss2 are increased in the female brain but suppressed in the male brain (11). In zebrafish, kiss1 is mainly expressed in the habenula (vHb) and kiss2 signals are distributed in the dorsal zone (Hd), the posterior tuberal nucleus (nPT), and the ventral (Hv) (22). The brain is regarded as the organ where kisspeptin genes primarily act, and the habenula mainly regulates circadian rhythm and stress response (23). The sexually dimorphic distribution of kiss-positive cells in the brain is reported in medaka and zebrafish (24, 25). Under reproductive conditions, more nucleus ventral tuberis (NVT) KiSS-1 neurons are observed in male medaka than females (24). In zebrafish, kiss2-positive cells are identified in the pituitary of females but not males (25).

The sexual reversal of hermaphroditic teleosts is associated with social stress and gonadal development (26). Acute and chronic stress with corticosterone decrease Kiss1 but increase Kiss1r expression in the medial preoptic area (mPOA) and the arcuate nucleus (ARC) of female rats (27). In the African cichlid fish (Astatotilapia burtoni), the male is a subordinate individual in the group, whose reproductive activity is inhibited and the expression of kiss1r is lower throughout brain (28). Recent studies have shown that kiss2 but not kiss1 is involved in the regulation of social stress and the gonad development in yellowtail clownfish (21). Social stress may directly act on the kisspeptin signal system via glucocorticoid and then participate in the regulation of gonadal differentiation and sexual reversal. However, the distribution of two kiss-expressing neurons in the brain of sexual reversal teleosts has been poorly studied.

The yellowtail clownfish is a protandrous hermaphroditic teleost whose sexual development can be regulated by its social status (29). In general, there is only one dominant female individual and one subdominant male with reproductive function in the group, while non-breeders are in the subordinate position. In the absence of female fish, the subdominant male individual will undergo sexual reversal and become the dominant female, and one non-breeder will become the mate of the subdominant male individual (30). Social sex determination of yellowtail clownfish is a specific reproductive phenomenon regulated by the social stress and HPG axis (31). The yellowtail clownfish is regarded as a suitable model to study the mechanism of social sex determination (32).

In the present study, we would explore the sexually dimorphic distribution of kiss1 and kiss2 mRNA in the brain of yellowtail clownfish using RNAscope in situ hybridization. The research on the distribution of kiss-expressing brain regions is the basis for elucidating the association between environmental cues and reproductive function.

Materials and methods

Animals

Sexually mature yellowtail clownfish were purchased from a local aquarium (Haikou city, Hainan, China). Fish were fed with commercial feeds twice a day (08:30 and 17:30 h) in culture system with circulating seawater for acclimatization. Water temperature was maintained at ranges from 26°C to 28°C and the photoperiod was a 12 h light:12 h darkness cycle. After acclimatization for a week, fish were anaesthetized with 0.05% MS222 (Sigma). The gonad and brain of each individual were fixed in Bouin's solution (Sigma) and 4% paraformaldehyde fix solution (Sigma), respectively.

This study protocol was reviewed and approved by Hainan University Institutional Animal Use and Care Committee, approval number HNUAUCC-2021-00014.

Histological sex identification and tissue preparation

The fixed gonad of each yellowtail clownfish was embedded in paraffin after ethanol dehydration and xylene transparency. The gonadal tissues were cut into 5 μm paraffin slices and stained with hematoxylin and eosin and then observed by microscope to determine sex of each individual (Fig. 1).

Figure 1
Figure 1

Histological slices were used to distinguish females and males according to the level of gonadal development in yellowtail clownfish. (A) The histological slices of the female gonad; (B) the histological slices of the male gonad. O1, oocytes in primary growth stage; O2, oocytes in cortical vesicle stage; O3, oocytes in vitellogenesis stage; SSC, secondary spermatocytes; S, spermatozoon. Scale = 200 μm.

Citation: Endocrine Connections 11, 8; 10.1530/EC-22-0136

All fixed brains were dehydrated through diethyl pyrocarbonate (DEPC)-treated PBS with 30% sucrose gradients and embedded in Tissue-Tek OCT (Sakura Finetechnical, Tokyo, Japan). The brain tissues of female (n  = 3) were cut into 10 μm sagittal slices on Superfrost® Plus Microscope Slides (Fisher Scientific). Depending on the distribution of kiss1and kiss2 signals in the female brain of yellowtail clownfish in sagittal planes (Fig. 2O), the brain tissues of female and male (n  = 3) were cut into 10 μm coronal slices (Fig. 3), respectively. The level of slices in the sagittal and coronal drawing view of yellowtail clownfish brain was separately shown in Figs 2P and 3. All manipulations were RNase-free.

Figure 2
Figure 2

The sagittal distribution of kiss1 and kiss2 mRNA in female yellowtail clownfish brain. (A, B, C and D) The kiss1-expressed brain regions in sagittal planes of the female brain; (E, F, G, H, I, J, K and L) the kiss2 expressed brain regions in sagittal planes of the female brain; (M) negative control; (N) positive control; (O) the level of the slices in the sagittal drawing view of female yellowtail clownfish brain; (P) the distribution of kiss1 and kiss2 in the female brain of yellowtail clownfish in sagittal planes. Tel, telencephalon; Vam, medial division of valvula cerebelli; CER, cerebellum; Hyp, hypothalamus; OT, optic tectum; DIE, diencephalon; MO, medulla oblongata; Hyp-NRLd, dorsal part of the nucleus of the lateral recess. Scale = 20 μm.

Citation: Endocrine Connections 11, 8; 10.1530/EC-22-0136

Figure 3
Figure 3

The distribution of kiss mRNA in the female and male brains of yellowtail clownfish in coronal planes. Black circles, the distribution of kiss mRNA in the female brain; open circles, the distribution of kiss mRNA in the male brain; black triangles, the distribution of kiss mRNA in the female brain; open triangles,the distribution of kiss mRNA in the male brain. CCe, corpus of the cerebellum; Cp, central posterior thalamic nucleus; DIp, lateral posterior part of the dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; Dm3, subdivision 3 of the medial dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NAPv, anterior periventricular nucleus; NDLIl, lateral part of the diffuse nucleus of the inferior lobe; NGp, posterior part of glomerular nucleus; NHd, dorsal habenular nucleus; NHv, ventral habenular nucleus; NLTI, inferior part of the lateral tuberal nucleus; NLTv, ventral part of the lateral tuberal nucleus; nMLF, nucleus of the medial longitudinal fasciculus; NPC, central pretectal nucleus; NPGa, anterior preglomerular nucleus; NPGc, commissural preglomerural nucleus; NPOav, anteroventral part of the parvocellular preoptic nucleus; NPOPc, parvocellular part of the parvocellular preoptic nucleus; NPT, posterior tuberal nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NRLv, ventral part of the nucleus of the lateral recess; NRP, nucleus of the posterior recces; LFB, lateral forebrain bundle; OC, optic chiasm; OT, optic tectum; PCo, posterior commissure; PGZ, periglomerular gray zone; PMgc, gigantocellular part of the magnocellular preoptic nucleus; PSp, parvocellular superficial pretectal nucleus; TEG, tegmentum; TLa, nucleus of the torus lateralis; TLo, torus longitudinalis; TPp, periventricular nucleus of the posterior tuberculum; VCe, valvula of the cerebellum; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VOT, ventral optic tract; VP, postcommissural part of the ventral telencephalon.

Citation: Endocrine Connections 11, 8; 10.1530/EC-22-0136

Fluorescent in situ hybridization

RNAscope probes were designed with reference to the kiss1 (GenBank No.: MK368701) and kiss2 (GenBank No.: MK368702) genes of yellowtail clownfish and listed in Table 1. Fluorescent in situ hybridization (FISH) was provided by RNAscope® Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics, Hayward, USA). Briefly, the cleared slices were incubated with hydrogen peroxide for 10 min at room temperature and then treated in boiling 1× RNAscope® Target Retrieval Reagents after washing with RNase-free water. The slices were washed with RNase-free water and ethanol to ensure complete drying at room temperature. RNAscope® Protease Reagents were dropped onto the slices and treated at 40°C for 30 min. After washing, the brain tissue slices of yellowtail clownfish were incubated with the probe solution in ACD HybEZ™ II Hybridization System (ACD Bio-Techne, USA) at 40°C for 2 h and washed twice in 1× RNAscope® wash buffer. Slices were sequentially immersed in AMP-1 and AMP-2 reagent at 40°C twice for 30 min each and finally immersed in AMP-3 reagent at 40°C twice for 15 min each. RNAscope® HRP-C1 signal was developed and employed TSA® Plus Cy3 (Perkin Elmer) to mark probe. All treated slices were incubated with DAPI for 30 sbefore being washed and cover coverslips with Prolong Gold Antifade (Thermo Fisher Scientific) mounting medium. Images were captured by fluorescence confocal microscopy (Nikon ECLIPSE Ti2) and analyzed on selected regions by NIS-Elements AR 5.30.02.

Table 1

Name of targets and catalog number of probes used for present study.
RNAscope-probe Cat No.
Acl-kiss1-C1 1044931-C1
Acl-kiss2-C1 1044941-C1
Aoc-actb2-C1 1045881-C1
Negative control probe-DapB 310043

Results

Distribution of kiss1 and kiss2 mRNA in the brain of yellowtail clownfish

kiss1 and kiss2 signals were detected in the sagittal planes of yellowtail clownfish brain (Fig. 2). In the female brain, the kiss1-positive signal was observed in the telencephalon (Tel), medial division of valvula cerebelli (Vam), cerebellum (CER), and hypothalamus (Hyp) (Fig. 2A, B, C and D). The kiss2 mRNA was the whole brain distributed and mainly expressed in Tel, optic tectum (OT), Vam, CER, diencephalon (DIE), medulla oblongata (MO), and Hyp (Fig. 2E, F, G, H, I, J, K and L). Based on the distribution of kiss1 and kiss2 signals in the female brain of yellowtail clownfish in sagittal planes (Fig. 2P), four coronal slices were selected for the next studies (Fig. 3).

Sexually dimorphic distribution of kiss1 mRNA in the brain of yellowtail clownfish

The distribution of kiss1 positive signals was marked in the coronal drawing view of yellowtail clownfish brain (Fig. 3). In females, the kiss1 signal was highly expressed at the dorsal habenular nucleus (NHd) of the habenula and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of hypothalamus, as well as minimally distributed in subdivision 3 of the medial dorsal telencephalic area (Dm3), subdivision 2 of the medial dorsal telencephalic area (Dm2), lateral posterior part of the dorsal telencephalic area (Dip), and posterior portion of the dorsal telencephalon (Dp) regions of the telencephalon (Fig. 4A, B, C, D, I, J and K). In males, the kiss1 mRNA was abundantly distributed at NHd in the habenula and low expressed at NDLIl of the hypothalamus. Compared with females, kiss1 was not detected in other brain regions of males (Fig. 4E, F, G, H, M, N and O).

Figure 4
Figure 4

The coronal distribution of kiss1 mRNA in female and male yellowtail clownfish brain. (A, B, C and D) and (I, J and K) The kiss1-expressed brain regions in coronal planes of the female brain; (E, F, G and H) and (M, N and O) the kiss1-expressed brain regions in coronal planes of the male brain; (L) negative control; (P) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NHd, dorsal habenular nucleus; CCe, corpus of the cerebellum; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.

Citation: Endocrine Connections 11, 8; 10.1530/EC-22-0136

Sexually dimorphic distribution of kiss2 mRNA in the brain of yellowtail clownfish

The distribution of the kiss2-positive signals was marked in the coronal drawing view of yellowtail clownfish brain (Fig. 3). In females, kiss2 transcripts were widely distributed in the telencephalon, midbrain, and hypothalamus, especially in the dorsal part of the nucleus of the lateral recess (NRLd) (Fig. 5A, B, C, D, E, F, M, N, O, P and Q). In males, kiss2-signaling molecules were found in the telencephalon, midbrain, and abundantly distributed at NRLd of the hypothalamus (Fig. 4G, H, I, J, K, L, S, T, U, V and W). The similar distribution of kiss2 mRNA was observed between males and females, whereas the stronger signal intensity of kiss2 was found in females than in males.

Figure 5
Figure 5

The coronal distribution of kiss2 mRNA in female and male yellowtail clownfish brain. (A, B, C, D, E and F) and (M, N, O, P and Q) the kiss2expressed brain regions in coronal planes of the female brain; (G, H, I, J, K and L) and (S, T, U, V and W) the kiss2-expressed brain regions in coronal planes of the male brain; (R) negative control; (X) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; OT, optic tectum; CCe, corpus of the cerebellum; TEG, tegmentum; TPp, periventricular nucleus of the posterior tuberculum; NGp, posterior part of glomerular nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.

Citation: Endocrine Connections 11, 8; 10.1530/EC-22-0136

Discussion

Sex determination of teleosts includes genotypic sex determination and environmental sex determination (ESD) (33). ESD has been deeply studied, but regulatory mechanisms in fish with the more complex social sex determination are still poorly understood (34, 35). Kisspeptin/GPR-54 signaling system is speculated as the key integrator between environmental cues and reproduction (2). In the present study, we investigated the distribution of kiss1 and kiss2 genes in the brain of both female and male yellowtail clownfish by RNAscope.

In mammals, kisspeptin neurons are mainly localized in the anteroventral periventricular (AVPV), the periventricular nucleus (PeN), and the arcuate (ARC) hypothalamic nucleus (1). Kisspeptin neurons show the wide distribution in brain of teleosts. In the zebrafish brain, kiss1 neurons are located in the ventromedial habenula and periventricular hypothalamic nucleus; thekiss2 neurons are distributed in the preoptic area (POA), midbasal hypothalamus, posterior tuberous nucleus, and periventricular hypothalamic nucleus (22, 36). In medaka, cells expressing kiss1 mRNA are mainly found in the habenula, hypothalamus, NVT, and nucleus posterioris periventricularis (NPPv). The distribution of medaka kiss2 neurons is similar to that in zebrafish (36). The distribution of kiss1 neurons in goldfish resembles that in zebrafish, and kiss2 mRNA is mainly expressed in the POA, nucleus lateralis tuberis (NLT), and nucleus recessus lateralis (NRL) (37, 38). In the present study, both kiss1- and kiss2-expressing cells were mainly distributed in the Tel, Vam, CER, and Hyp regions, while kiss2 signals were detected in the OT, DIE, and MO regions compared with kiss1. The kiss2 mRNA is more widely distributed in the brain of yellowtail clownfish than kiss1 mRNA. Moreover, kiss1 showed a high-intensity signal in NHd of the habenula and kiss2 was highly expressed at the NRLd of the hypothalamus. In African clawed frog (Xenopus Laevis), kiss gene signals are found in the ventral hypothalamus (VH), but kiss2 has more excess expression in the POA than kiss1 (36). The hypothalamus is considered a region of upstream regulation of the reproductive axis. The habenula, involved in behavioral responses related to pain, stress, anxiety and sleep, has the most conserved structure in the brain of vertebrates and is the main region of kiss1 distribution in teleosts such as zebrafish, medaka, goldfish, and European seabass (Dicentrarchus labrax) (22, 24, 38, 39, 40). Thus, yellowtail clownfish habenular kiss1 may be related to environmental and metabolic signals. In addition, kiss1r is detected in GnRH neurons of tilapia, suggesting that Kiss1 has a potential role in regulating reproduction (41).

The distribution of kiss mRNA is sexually dimorphic. In the NVT of medaka, males have a greater number of kiss1 neurons than females (24). Yellowtail clownfish kiss2 exhibited stronger signals in NRLd of the hypothalamus of the female than the male. Furthermore, our results showed that the kiss1 mRNA has a broader distribution pattern in females than in males. In red seabream (Pagrus major), kiss2 mRNA is mainly found in the NRLd and NRLv parts of hypothalamic nucleus recessi lateralis, and it has high expression in mature males compared with the male after spawning (42). In European seabass, kiss2r mRNA is detected in GnRH neurons (22). Moreover, kisspeptin-2 is more effective in regulating gonadotropin synthesis compared to kisspeptin-1 in zebrafish and medaka (36). Therefore, kiss2 might be associated with reproductive function.

Briefly, kiss1 is more widely distributed in females, and kiss2 is less abundant in males than females, implying that kiss1 and kiss2 might have different functions between sexuality and social status, and the lack of kiss2 mRNA leads to the delay of gonadal development. The previous studies showed that kiss1 and kiss2 show different expression patterns in yellowtail clownfish individuals under different social statuses, and kiss2 is considered to be the key regulatory gene in reproductive function (21). In goldfish, the GRE domain is found in the promoter region of kiss gene, suggesting that kisspeptin may be regulated by glucocorticoid receptor (GR) (43). It is reported that the ventromedial hypothalamic nucleus (VMH) has steroidogenic factor 1 (SF1; also known as Nr5a1) neurons, suggesting that glucocorticoids are associated with kisspeptin neurons in the hypothalamic region, especially kiss2 neurons (44). Moreover, different social status individuals with divergent cortisol levels are observed in Nile tilapia (45). Our previous study showed that GR2 is more sensitive to cortisol than GR1 in yellowtail clownfish (32). GR genes also show the sexually dimorphic expression in the brain of medaka, in which GR has high expression in several preoptic and thalamic nuclei of females (46).

In the present study, kiss2 had higher positive signals in primary brain nuclei, which may suggest that kiss genes have disparate functions among different brain regions. Furthermore, only one kiss gene (kiss2) is reported in Nile tilapia and puffer fish (Takifugu niphobles) (47, 48). Therefore, kiss2 might have an important role in reproductive function, whereas kiss1 may be involved in sensing environmental signals and metabolism (22). The results of the present research further support this hypothesis.

Conclusion

Sexually dimorphic distribution of kiss genes in the brain of yellowtail clownfish is studied. The kiss1 mRNA had wider and stronger signal intensity in female individuals than in males. The distribution of the kiss2-positive brain region was similar in both females and males, but the signal intensity was stronger in females. In our results, kiss1/kiss2 signals were detected in NDLIl and NRLd of hypothalamus implicating the possible involvement of kiss genes in reproductive regulation. Moreover, the kiss1 signals detected in habenula suggest that kiss1 may be associated with environmental and metabolic signals, such as social stress, pain, and anxiety. At last, kiss genes involved in environmental cues and reproductive function may be key regulators of sex reversed fish with ESD.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by National Natural Science Foundation of China (NSFC Grant No. 31760759).

Data availability statement

The data sets that were analyzed during the current study are available from the corresponding author on reasonable request.

Author contribution statement

Zhang Yan-yu, Zhang Xian, and Wang Qian contributed to the study design. Zhang Xian, Bu Shao-yang, Zhang Wei-wei, Li Tian-xiu, Zheng De-cai, and Huang Ze-xiang contributed to the acquisition of data. Zhang Yan-yu, Zhang Xian, and Bu Shao-yang performed statistical analyses. Zhang Yan-yu and Wang Qian drafted the manuscript. Zhang Yan-yu, Zhang Xian, Bu Shao-yang, Zhang Wei-wei, Li Tian-xiu, Zheng De-cai, Huang Ze-xiang, and Wang Qian contributed to data interpretation, provided critical revisions, and approved the final version of the manuscript.

Acknowledgements

The authors thank Dr Tang Chaorong and Dr Zhang Yi from Laboratory of Rubber Production Biology of Hainan University for help with the use of fluorescence confocal microscopy.

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Online ISSN:
2049-3614
Copyright:
© The authors 2022
Article ID:
e220136
Article Type:
Research Article
Received Date:
10 Jun 2022
Accepted Date:
27 Jun 2022
Online Publication Date:
25 Jul 2022
Keywords:
kisspeptin; social stress; sexually dimorphic distribution; yellowtail clownfish; RNAscope
  • Figure 1
    View in gallery
    Figure 1

    Histological slices were used to distinguish females and males according to the level of gonadal development in yellowtail clownfish. (A) The histological slices of the female gonad; (B) the histological slices of the male gonad. O1, oocytes in primary growth stage; O2, oocytes in cortical vesicle stage; O3, oocytes in vitellogenesis stage; SSC, secondary spermatocytes; S, spermatozoon. Scale = 200 μm.

  • Figure 2
    View in gallery
    Figure 2

    The sagittal distribution of kiss1 and kiss2 mRNA in female yellowtail clownfish brain. (A, B, C and D) The kiss1-expressed brain regions in sagittal planes of the female brain; (E, F, G, H, I, J, K and L) the kiss2 expressed brain regions in sagittal planes of the female brain; (M) negative control; (N) positive control; (O) the level of the slices in the sagittal drawing view of female yellowtail clownfish brain; (P) the distribution of kiss1 and kiss2 in the female brain of yellowtail clownfish in sagittal planes. Tel, telencephalon; Vam, medial division of valvula cerebelli; CER, cerebellum; Hyp, hypothalamus; OT, optic tectum; DIE, diencephalon; MO, medulla oblongata; Hyp-NRLd, dorsal part of the nucleus of the lateral recess. Scale = 20 μm.

  • Figure 3
    View in gallery
    Figure 3

    The distribution of kiss mRNA in the female and male brains of yellowtail clownfish in coronal planes. Black circles, the distribution of kiss mRNA in the female brain; open circles, the distribution of kiss mRNA in the male brain; black triangles, the distribution of kiss mRNA in the female brain; open triangles,the distribution of kiss mRNA in the male brain. CCe, corpus of the cerebellum; Cp, central posterior thalamic nucleus; DIp, lateral posterior part of the dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; Dm3, subdivision 3 of the medial dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NAPv, anterior periventricular nucleus; NDLIl, lateral part of the diffuse nucleus of the inferior lobe; NGp, posterior part of glomerular nucleus; NHd, dorsal habenular nucleus; NHv, ventral habenular nucleus; NLTI, inferior part of the lateral tuberal nucleus; NLTv, ventral part of the lateral tuberal nucleus; nMLF, nucleus of the medial longitudinal fasciculus; NPC, central pretectal nucleus; NPGa, anterior preglomerular nucleus; NPGc, commissural preglomerural nucleus; NPOav, anteroventral part of the parvocellular preoptic nucleus; NPOPc, parvocellular part of the parvocellular preoptic nucleus; NPT, posterior tuberal nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NRLv, ventral part of the nucleus of the lateral recess; NRP, nucleus of the posterior recces; LFB, lateral forebrain bundle; OC, optic chiasm; OT, optic tectum; PCo, posterior commissure; PGZ, periglomerular gray zone; PMgc, gigantocellular part of the magnocellular preoptic nucleus; PSp, parvocellular superficial pretectal nucleus; TEG, tegmentum; TLa, nucleus of the torus lateralis; TLo, torus longitudinalis; TPp, periventricular nucleus of the posterior tuberculum; VCe, valvula of the cerebellum; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VOT, ventral optic tract; VP, postcommissural part of the ventral telencephalon.

  • Figure 4
    View in gallery
    Figure 4

    The coronal distribution of kiss1 mRNA in female and male yellowtail clownfish brain. (A, B, C and D) and (I, J and K) The kiss1-expressed brain regions in coronal planes of the female brain; (E, F, G and H) and (M, N and O) the kiss1-expressed brain regions in coronal planes of the male brain; (L) negative control; (P) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NHd, dorsal habenular nucleus; CCe, corpus of the cerebellum; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.

  • Figure 5
    View in gallery
    Figure 5

    The coronal distribution of kiss2 mRNA in female and male yellowtail clownfish brain. (A, B, C, D, E and F) and (M, N, O, P and Q) the kiss2expressed brain regions in coronal planes of the female brain; (G, H, I, J, K and L) and (S, T, U, V and W) the kiss2-expressed brain regions in coronal planes of the male brain; (R) negative control; (X) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; OT, optic tectum; CCe, corpus of the cerebellum; TEG, tegmentum; TPp, periventricular nucleus of the posterior tuberculum; NGp, posterior part of glomerular nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.

Figure 1

Histological slices were used to distinguish females and males according to the level of gonadal development in yellowtail clownfish. (A) The histological slices of the female gonad; (B) the histological slices of the male gonad. O1, oocytes in primary growth stage; O2, oocytes in cortical vesicle stage; O3, oocytes in vitellogenesis stage; SSC, secondary spermatocytes; S, spermatozoon. Scale = 200 μm.
Figure 2

The sagittal distribution of kiss1 and kiss2 mRNA in female yellowtail clownfish brain. (A, B, C and D) The kiss1-expressed brain regions in sagittal planes of the female brain; (E, F, G, H, I, J, K and L) the kiss2 expressed brain regions in sagittal planes of the female brain; (M) negative control; (N) positive control; (O) the level of the slices in the sagittal drawing view of female yellowtail clownfish brain; (P) the distribution of kiss1 and kiss2 in the female brain of yellowtail clownfish in sagittal planes. Tel, telencephalon; Vam, medial division of valvula cerebelli; CER, cerebellum; Hyp, hypothalamus; OT, optic tectum; DIE, diencephalon; MO, medulla oblongata; Hyp-NRLd, dorsal part of the nucleus of the lateral recess. Scale = 20 μm.
Figure 3

The distribution of kiss mRNA in the female and male brains of yellowtail clownfish in coronal planes. Black circles, the distribution of kiss mRNA in the female brain; open circles, the distribution of kiss mRNA in the male brain; black triangles, the distribution of kiss mRNA in the female brain; open triangles,the distribution of kiss mRNA in the male brain. CCe, corpus of the cerebellum; Cp, central posterior thalamic nucleus; DIp, lateral posterior part of the dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; Dm3, subdivision 3 of the medial dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NAPv, anterior periventricular nucleus; NDLIl, lateral part of the diffuse nucleus of the inferior lobe; NGp, posterior part of glomerular nucleus; NHd, dorsal habenular nucleus; NHv, ventral habenular nucleus; NLTI, inferior part of the lateral tuberal nucleus; NLTv, ventral part of the lateral tuberal nucleus; nMLF, nucleus of the medial longitudinal fasciculus; NPC, central pretectal nucleus; NPGa, anterior preglomerular nucleus; NPGc, commissural preglomerural nucleus; NPOav, anteroventral part of the parvocellular preoptic nucleus; NPOPc, parvocellular part of the parvocellular preoptic nucleus; NPT, posterior tuberal nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NRLv, ventral part of the nucleus of the lateral recess; NRP, nucleus of the posterior recces; LFB, lateral forebrain bundle; OC, optic chiasm; OT, optic tectum; PCo, posterior commissure; PGZ, periglomerular gray zone; PMgc, gigantocellular part of the magnocellular preoptic nucleus; PSp, parvocellular superficial pretectal nucleus; TEG, tegmentum; TLa, nucleus of the torus lateralis; TLo, torus longitudinalis; TPp, periventricular nucleus of the posterior tuberculum; VCe, valvula of the cerebellum; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VOT, ventral optic tract; VP, postcommissural part of the ventral telencephalon.
Figure 4

The coronal distribution of kiss1 mRNA in female and male yellowtail clownfish brain. (A, B, C and D) and (I, J and K) The kiss1-expressed brain regions in coronal planes of the female brain; (E, F, G and H) and (M, N and O) the kiss1-expressed brain regions in coronal planes of the male brain; (L) negative control; (P) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; NHd, dorsal habenular nucleus; CCe, corpus of the cerebellum; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.
Figure 5

The coronal distribution of kiss2 mRNA in female and male yellowtail clownfish brain. (A, B, C, D, E and F) and (M, N, O, P and Q) the kiss2expressed brain regions in coronal planes of the female brain; (G, H, I, J, K and L) and (S, T, U, V and W) the kiss2-expressed brain regions in coronal planes of the male brain; (R) negative control; (X) positive control. Dm3, subdivision 3 of the medial dorsal telencephalic area; Dm2, subdivision 2 of the medial dorsal telencephalic area; DIp, lateral posterior part of the dorsal telencephalic area; Dp, posterior portion of the dorsal telencephalon; OT, optic tectum; CCe, corpus of the cerebellum; TEG, tegmentum; TPp, periventricular nucleus of the posterior tuberculum; NGp, posterior part of glomerular nucleus; NRLd, dorsal part of the nucleus of the lateral recess; NDLIl, lateral part of the diffuse nucleus of the inferior lobe. Scale = 20 μm.
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    Guo Y, Wang Q, Li G, He M, Tang H, Zhang H, Yang X, Liu X, Lin H. Molecular mechanism of feedback regulation of 17β-estradiol on two kiss genes in the protogynous orange-spotted grouper (Epinephelus coioides). Molecular Reproduction and Development 2017 84 495–507. (https://doi.org/10.1002/mrd.22800)

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