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Genome-wide identification of Gα family in grass carp (Ctenopharyngodon idella) and reproductive regulation functional characteristics of Cignaq

BMC Genomics volume 25, Article number: 800 (2024) Cite this article

Abstract

Background

The Gα family plays a crucial role in the complex reproductive regulatory network of teleosts. However, the characterization and function of Gα family members, especially Gαq, remain poorly understood in teleosts. To analyze the characterization, expression, and function of grass carp (Ctenopharyngodon idella) Gαq, we identified the Gα family members in grass carp genome, and analyzed the expression, distribution, and signal transduction of Gαq/gnaq. We also explored the role of Gαq in the reproductive regulation of grass carp.

Results

Our results showed that the grass carp genome contains 27 Gα genes with 46 isoforms, which are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The expression level of Cignaq in the testis was the highest and significantly higher than in other tissues, followed by the hypothalamus and brain. The luteinizing hormone receptor (LHR) was mainly localized to the nucleus in grass carp oocytes, with signals also present in follicular cells. In contrast, Gαq signal was mainly found in the cytoplasm of oocytes, with no signal in follicular cells. In the testis, Gαq and LHR were co-localized in the cytoplasm. Furthermore, the grass carp Gαq recombinant protein significantly promoted Cipgr expression.

Conclusions

These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation of teleosts.

Peer Review reports

Background

Reproduction is a crucial biological process in the evolution of species, resulting in the creation of new offspring from their parents [1]. In mammals, this process is driven by the gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR), which regulate the production of gonadotropins [2, 3]. However, in teleosts, the regulation of reproduction may involve multiple factors that act in parallel and interact with GnRH. These include neurotransmitters and neuropeptides such as dopamine [4,5,6], gonadotropin inhibitory hormone (GnIH) [7, 8], γ- aminobutyric acid (GABA) [9, 10], serotonin [11], neuropeptide Y [12], spexin [13], neurokinin B [14], and neurosecretonin [15, 16].

G protein-coupled receptors (GPCRs) and G proteins are one of the largest transmembrane signaling systems that participate in various essential physiological processes. They can bind to a wide variety of ligands, including hormones, proteins, peptides, amino acids, lipids, nucleotides, and xenobiotics [17, 18]. GPCRs are widely distributed, with more than 800 identified in mammalian [19]. G proteins, composed of α, β, and γ subunits, act as molecular switches in many GPCRs signaling pathways [20]. There are 16 different Gα genes with at least 21 isoforms identified in the human genome, which can be grouped into four functional subfamilies (Gαs, Gαi, Gαq, and Gα12) based on sequence similarity [21]. These distinct Gα subfamilies specify both GPCR interactions and the transduction of downstream signaling events [22]. The Gαs subfamily can trigger adenylate cyclase (AC), which increases the intracellular cyclic adenosine monophosphate (cAMP) level. This further activates protein kinase A (PKA) and downstream signal cascades [23, 24]. In contrast, activation of Gαi leads to AC inhibition and a decrease in cAMP levels [25, 26]. However, some members of the Gαi subfamily, such as Gαt1and Gαt2, can also specifically activate downstream pathways [27]. The Gα12 subfamily can activate the GTPase activity of small G proteins, which is the main GPCR-G protein pathway to activate Rho [28,29,30,31]. The Gαq subfamily can activate phospholipase C β (PLCβ), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphonate (PIP2) to produce diacylglycerol (DAG) and inositol trisphosphate (IP3) [32]. DAG activates protein kinase C (PKC), and IP3 acts on Ca2+ channels in the endoplasmic reticulum, inducing Ca2+ release into the cytoplasm and further transmitting signals downstream [33]. These signal pathways mediated by the Gα family constitute a complex regulatory network that regulates system functions, such as embryonic and gonadal development, and organismal homeostasis [34, 35]. The Gα family also plays a crucial role in the complex reproductive regulatory network of teleosts. For instance, dopamine receptors trigger the Gαi subfamily to transmit information into the cell, inhibiting the synthesis and secretion of gonadotropins in tilapia (Oreochromis mossambicus) [36]. Neuropeptide Y can stimulate luteinizing hormone (LH) secretion through the Gαi-mediated signaling pathway in catfish (Clarias batrachus) [37].

The Gαq protein, coded by the gnaq gene, serves as a molecular switch in certain reproductive signaling pathways. It plays a crucial role in regulating multiple pathways involved in the reproductive axis, including the maintenance of GnRH synthesis and secretion through the kisspeptin receptor [38,39,40]. Additionally, Gαq can activate the GnRH receptor signaling pathway, leading to the synthesis and secretion of LH and FSH [41]. Furthermore, LH can simulate the expression of Pgr and promote follicle rupture through Gαq signaling [42]. Our previous research has shown that gnaq is widely expressed in zebrafish tissues, with the highest expression in the hypothalamus during the embryonic development stage after 72 h post-fertilization. This suggests that gnaq/Gαq may play a significant role in fish reproduction [43]. However, due to the vast number of teleost species, the characterization and function of the Gα family, particularly Gαq, remain poorly understood in teleosts.

In this study, we aimed to identify the members of the Gα family in the genome of grass carp (Ctenopharyngodon idella). Additionally, we analyzed the expression, distribution, and signal transduction of Gαq/gnaq, and conducted a preliminary investigation into the role of Gαq in the reproductive regulation of grass carp. This study provides initial evidence for understanding the involvement of Gαq in reproductive regulation in teleost fish.

Methods

Identification of Gα family members in the grass carp genome

The genome data for grass carp (NCBI RefSeq assembly ID: GCF_019924925.1) was obtained from a public database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_019924925.1/) [44]. HMM models of the Gα domain (PF00503) were searched for in the Pfam database (https://www.ebi.ac.uk/interpro/), and the Gα genes were identified in the grass carp genome using TBtools v1.1061 [45] with the simple search function. The protein sequences of the human Gα family [46] were compared to the grass carp protein data using a multi-sequence alignment algorithm. The intersection of the two result files was extracted and submitted to the SMART (http://smart.embl-heidelberg.de/) for domain analysis. Redundant sequences were manually filtered out, and the grass carp Gα genes were identified based on the presence of the conserved Gα domain. The physicochemical properties of the Gα family members were analyzed, and their subcellular localization was predicted using the online website WoLF PSORT (https://wolfpsort.hgc.jp/).

Dendrogram construction of Gα family members, domains, motifs, and gene structure

A neighbor-joining dendrogram of the Gα family was constructed using MEGA 7, based on amino acid sequences. This was done with 1000 bootstraps, as previously described [47]. The domain architectures of Gα family members were collected from CDD [48]. The protein sequences of Gα family members were submitted to the MEME Suite 5.5.2 (https://meme-suite.org/meme/tools/meme) with a parameter of 10 for selecting the number of motifs [49]. These results were visualized using TBtools and further refined using Adobe Illustrator 2022 (version 26.0).

Chromosomal localization, gene duplication and collinearity analysis

The information on chromosomal localization and of Gα family members was retrieved from the grass carp genomic database and used to construct a genetic map using TBtools software. Putative duplication events were detected within the Gα family, with tandem duplications identified as two proteins with a similarity of over 40% and separated by four or fewer gene loci. Duplications separated by more than five genes were classified as segmental duplications [50]. The whole genome annotation files for zebrafish (GRCz11.109) were downloaded from the Ensembl Animal Genome database 109 (https://ftp.ensembl.org/pub/release-109/gff3/danio_rerio/) and analyzed for genomic collinearity blocks using TBtools software. The rates of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) for duplicate genes were calculated using TBtools software, following previous methods, to investigate the selective pressures on Gα genes [51]. The grand average of hydropathicity (GRAVY) for Gα proteins was determined using the GRAVY Calculator (https://www.gravy-calculator.de/index.php?page=file). Proteins with a GRAVY value < -5 indicate high hydrophilicity, suggesting they are hydrophilic proteins. Proteins with a GRAVY value between -0.5 and 0.5 are considered amphiphilic, while those with a GRAVY value > 0.5 are relatively hydrophobic, indicating they are hydrophobic proteins.

Expression analysis of Gα gene family members in hypothalamus–pituitary–gonadal (HPG) axis tissues of grass carp

Samples of the hypothalamus, pituitary, and gonad were collected from fifteen female grass carp individuals in Wulong Fishing Ground, Liuyang City, Hunan Province, China (Table S1). The grass carp samples were purchased from the privately farm with the informed consent of the owners for research purposes. The animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Human Agricultural University (HAU). Prior to sampling, the grass carp samples were anesthetized by soaking with 100 mg/L tricaine methanesulfonate (MS-222) for 5 min. Samples of five individuals were mixed as one sample for transcriptome sequencing. The transcriptome of each mixed sample was sequenced using the Novaseq 6000 sequencing platform (Illumina, USA). The grass carp HPG transcriptome data (not yet published) were analyzed using BLAST to identify the coding sequences of Gα family members. The identified Gα family members were then searched for in the transcriptome data. The reads per kilobase per million mapped reads (RPKM) for the Gα family members were obtained from the transcriptome data and normalized using TBtools to produce a heatmap.

Sample collection, total RNA extraction and RT-qPCR analysis

The grass carp used in the experiment were cultured at the Cultivation Base of Hunan Agricultural University (Table S1). The samples were owned by our institution and the animal procedures were reviewed and approved by the IACUC of HAU. Prior to the experiment, the grass carp were removed from the pond and placed in a circulating water aquaculture system in the laboratory for two weeks, with a constant water temperature of 28 °C. Prior to sampling, the grass carp samples were anesthetized by soaking with 100 mg/L tricaine methanesulfonate (MS-222) for 5 min. Samples of the brains, heart, gills, liver, spleen, kidney, ovary, and testis were obtained from three individual grass carp, while samples of the hypothalamus and pituitary were obtained from ten individual grass carp for cloning and gnaq mRNA profiling. Tissue total RNA was extracted using an RNA-easy Isolation Reagent (Vazyme, China), and cDNA was synthesized using a Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States).

The RT-qPCR was conducted according to the previous method [43]. Moreover, the total RNA was extracted from hemocytes for analysis of gnaq expression level. β-Actin and ef1α genes were used as reference gene (Table S2). Each experiment was replicated three times and the data were analyzed using the 2−ΔΔCt method.

Preparation of grass carp recombinant Gαq protein (reGαq)

To obtain grass carp reGαq, the PCR product of Cignaq was ligated into the pGEX-4 T-1 expression vector. The recombinant plasmid was transformed into Escherichia coli BL21. Then, the bacteria were incubated in LB medium with 1 mM IPTG for 16 h at 16 °C with 180 rpm shaking. After induction, the bacteria were sonicated to lyse them and the supernatant was harvested. An Ni–NTA Sepharose column was used to purify the recombinant GST-tagged Gαq proteins. The purified reGαq was dialyzed three times against Tris-buffered saline (50 mM Tris–HCl, 150 mM NaCl, pH 7.4) at 4 °C. The concentration was determined using a BCA assay kit (Sigma-Aldrich, USA).

GnRH and gonadotropin hormone detection

The serum and hemocytes were sampled from female grass carp collected from Tianjiahu Fishing Ground in Huarong County (Yueyang, Hunan, China) before administer an oxytocin and after ovulation (Table S1). The grass carp samples were purchased from the privately farm with the informed consent of the owners for research purposes. The animal procedures were reviewed and approved by the IACUC of HAU. Prior to sampling, the grass carp samples were anesthetized by soaking with 100 mg/L tricaine methanesulfonate (MS-222) for 5 min. The GnRH and gonadotropin hormone were detected in the serum using fish ELISA reagent (ZCIBIO, China), according to the manufacturer’s instructions.

Immunohistochemistry and immunofluorescence

The immunohistochemistry and immunofluorescence procedures were carried out according to previously published methods [43, 52]. The testis, ovary, pituitary, and brain tissues of grass carp were fixed in 4% paraformaldehyde at 4 °C for 24 h, dehydrated in a series of alcohol solutions, cleared in toluene, and embedded in paraffin wax. Transverse sections of 5 μm thickness were then dried at 60 °C for 2 h and placed on glycerin-coated slides. After dewaxing, the sections were rehydrated and washed with phosphate buffered solution (PBS), followed by incubation with 3% hydrogen peroxide in PBS for 30 min to block endogenous peroxidase activity. For immunofluorescence, the sections were boiled in Citrate buffer for 15 min at 95 °C and allowed to cool to 25 °C. They were then washed three times with PBS for 5 min each. Next, the sections were incubated overnight at 4 °C with Gαq antibody (ABclonal, Wuhan, China). After three washes with PBST, the sections were incubated with goat-anti-rat IgG (Abcam, Britain) at a dilution of 1:5,000 for 45 min at 37 °C. The sections were then treated with DAB chromogenic solution (Maxim, China) for 5 min, followed by a 1-min application of hematoxylin (BaSO, China) to stain the nuclei. For immunofluorescence, DAPI was used to stain the nuclei at a dilution of 1:1,000 for 5 min. The sections were washed three times with PBS for 5 min each and then sealed with an anti-quenching fluorescent sealing agent. Finally, the sections were observed and photographed using an Olympus microscope (Olympus, Japan).

Cell culture

The assays were conducted using the previous method with slightly modifications [47]. The grass carp ovarian (GCO) cell line was obtained through passage culture in our study group. The GCO cells were cultured in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 28 °C with 5% CO2 and Medium DMEM (Gibco, Grand Island, NY, USA) liquid medium containing a 1% penicillin–streptomycin mixture and 10% fetal bovine serum. Once the cells covered 80% of the bottom of the culture flask (Corning, NY, USA), they were detached using trypsin and transferred into 6-well plates (Corning, NY, USA) for transfection and incubation with hCG. Total RNA was extracted from the cell samples for gene mRNA profile analysis.

Data analysis

The data were presented as mean ± standard error. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 25) software for Shapiro–Wilk normality test, Bartlett test of homogeneity of variances, one-way analysis of variance (ANOVA), and Kruskal–Wallis rank sum test. If each group of data conformed to normal distribution and variance homogeneity, one-way ANOVA was used to test, otherwise Kruskal–Wallis rank sum test was used. Results with a p-value of less than 0.05 were considered statistically significant. GraphPad Prism 7 software was used for creating graphs.

Results

Genome-wide identification of Gα family in grass carp

Twenty-seven Gα genes with 46 isoforms were identified in the grass carp genome, with lengths ranging from 350 to 491 amino acids (Table 1 and S3). Among them, Cignav1 did not have a homologous gene in the mammalian genome. The molecular weights (MWs) of the Gα family proteins ranged from 39.83 to 56.17 kDa. The isoelectric points of the 14 acidic and 13 alkaline proteins ranged from 5.18 to 6.99 and from 7.6 to 9.53, respectively. Subcellular localization prediction indicated that Gα subunits were mostly located in the cytoplasm. The instability index of only five proteins was less than 40, indicating a relatively stable protein structure. The GRAVY results of the Gα proteins showed that three were hydrophilic (Cignas, Cignal, and Cignal2), while the others had GRAVY values ranging from -0.5 to 0.5 (Table 1), indicating that they are amphiphilic proteins.

Table 1 Overall information for Gα family
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Dendrogram and structural features of Gα family

According to the neighbor-joining dendrogram, 27 grass carp Gα family genes were divided into four subfamilies: Gαs (Cignas, Cignal, and Cignal2), Gαi/o (Cignai, Cignai1, Cignaia, Cignai2a, Cignai2b, Cignat1, Cignat2, Cignaoa, Cignaoa-like, Cignaob, Cignaz, and Cignav1), Gαq/11 (Cignaq, Cigna11, Cigna11-like, Cigna11a, Cigna11b, Cigna14, Cigna14-like, and Cigna15.1), and Gα12/13 (Cigna12, Cigna12a, Cigna13a, and Cigna13b) (Fig. 1A and S1). The gene structure analysis of the Gα family revealed that the position and length of the UTR region and introns varied greatly within the same subfamily, while the number and length of exons were similar. The number of exons also varied greatly among the different subfamilies, with Gα12/13, Gαq/11, Gαi/o, and Gαs subfamily members containing four, seven, 8–9, and 11–12 exons, respectively (Fig. 1B). The prediction of 10 conserved motifs of Gα family members showed that all Gα subunits contained motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, and motif 7 (Fig. 1C). Further analysis of the conservative domains revealed that members of the Gα family only contained the G-alpha domain, indicating that the Gα subunit family was relatively conservative (Fig. S2).

Fig. 1
figure 1

Schematic diagram of exon–intron structure and motifs of the Gα family. A Dendrogram of the Gα gene family was divided into four groups: Gαi/o (orange), Gαs (blue), Gαq (red), and Gα12/13 (green) subfamilies. B Exon–intron structure with the scale below representing the length of the base. C Motif structure diagram of the Ga family with the bottom scale indicating the length of amino acids

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Chromosomal location, gene duplication, and syntenic analysis

The 27 Gα family genes were distributed across 14 chromosomes, which one gene on the first, second, fourth, seventh, eleventh, twelfth, eighteenth, and twenty-fourth chromosomes, three genes on the third, sixth, and eighth chromosome, two genes on the fifth and twenty-third chromosomes, and six genes on the tenth chromosome (Fig. 2A). Additionally, a gene cluster was identified on the tenth chromosome, with all genes belonging to the Gαq/11 subfamily. These findings suggest that the Gα family in grass carp has undergone significant expansion compared to the human Gα family.

Fig. 2
figure 2

Chromosomal location and gene duplication (A), and syntenic analysis (B) of the grass carp Gα family

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The gene families evolved through genome-wide duplication, segmental duplication, or tandem duplication, and gene diversification occurred after these duplication events [53,54,55,56]. The duplication patterns (tandem and segmental duplication) were identified to elucidate the mechanism underlying the expansion of the Gα family in grass carp. The results indicated that three segmental and eight tandem duplication events were detected (Fig. 2A and Table 2).

Table 2 Divergences between paralogous Gα gene pairs in grass carp
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To further understand the evolutionary forces at play in the grass carp Gα family, Ka and Ks values were calculated for the duplication pairs in the family (Table 2). The Ks value of the Cignaq/Cigna14 gene pair could not be calculated due to the high sequence dispersion value (pS >  = 0.75). The Ka/Ks values of the other gene pairs ranged from 0.081 to 0.601, indicating that repetitive genes underwent intense purification selection pressure, and the duplication-producing gene had evolved enormously while maintaining its functional stability.

Synteny analysis between the grass carp and zebrafish genomes revealed 20 pairs of orthologous gene pairs, among which Cigna11, Cigna11a, Cigna11-like, Cigna14-like, Cignao-like, Cignat2, and Cignav1 did not have corresponding homologous genes in zebrafish, indicating that the grass carp Gα family had higher homology than zebrafish (Fig. 2B).

Tissue expression profile of grass carp Gα family on HPG axis

To analyze the expression pattern of Gα family members on the grass carp HPG axis, the FPKM values of these members were extracted from the grass carp HPG axis transcriptome (Table S4). Only 15 Gα family members were detected in the transcriptome, including Cignaia, Cigna13b, Cigna12a, Cignav1, Cignai1, Cigna11b, Cignai13a, Cignaz, Cigna11a, Cignaq, Cignal, Cignal2, Cignas, Cignai2b, and Cignaoa. Among these, Cignaoa, Cignaq, Cignai2b, Cignal, Cigna11b, and Cignai1 were highly expressed in the hypothalamus, while Cignal2, Cignas, Cignaz, Cignaia, Cignav1, Cigna13b, Cigna12a, and Cigna13a were highly expressed in the gonads (Fig. 3A and S3). No genes were found to be significantly highly or lowly expressed in the pituitary compared to those in the hypothalamus and gonads (Fig. S3).

Fig. 3
figure 3

Tissue expression profile of the Gα family on the HPG axis in grass carp transcriptomes (A) and tissue expression levels of grass carp gnaq (B). H01-H03 represent three biological repeats of grass carp hypothalamus; G01-G03 represent three biological repeats of grass carp gonads; P01-P03 represent three biological repeats of grass carp pituitary. Different lowercase letters above the bars indicate significant differences between data

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RT-qPCR results showed that the expressions levels of Cignaq varied among different tissues in grass carp. The highest expression level of Cignaq was found in the testis, significantly higher than in other tissues (P < 0.05), followed by the hypothalamus and brain (Fig. 3B).

Analysis of Cignaq expression characteristics in grass carp

The concentrations of GnRH and gonadotropin, as well as the expression level of Cignaq, were examined in the female parent of grass carp before and after ovulation. While there was no significant difference in the concentrations of LH and FSH before and after ovulation (P > 0.05; Fig. 4B and C), the concentration of GnRH in the blood before ovulation was significantly lower than that after ovulation (P < 0.05; Fig. 4A). Additionally, the expression level of Cignaq mRNA was found to be opposite (P < 0.01; Fig. 4D).

Fig. 4
figure 4

mRNA levels of GnRH, LH, FSH, and gnaq in grass carp before and during ovulation. A Expression level of GnRH in blood; B Expression level of LH in blood; C Expression level of FSH in blood; D Expression level of gnaq in grass carp. The error bar represents the mean ± SEM (n = 5). * P < 0.05; ** P < 0.01

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Localization of Gαq in grass carp brain–pituitary–gonadal axis

The brain of the grass carp was divided into six parts: olfactory bulb, telencephalon, optic tectum, hypothalamus, cerebellum, and medulla oblongata [57]. In order to investigate the distribution of Gαq protein in the grass carp brain, this study was conducted from three different perspectives. The results showed that Gαq protein was not present in the granular layer of the cerebellum, but was highly expressed in the telencephalon, optic tectum, hypothalamus, hindbrain, neurohypophysis, and adenohypophysis (Fig. 5 and S4). This suggests that Gαq protein plays a role in regulating various physiological activities.

Fig. 5
figure 5

Distribution of Gαq protein in the brain and pituitary of grass carp. A-C Immunohistochemistry of Gαq protein in grass carp brain in apical view; D-F Immunohistochemistry of Gαq protein in grass carp brain longitudinal sections; G-K Immunohistochemistry of Gαq protein in grass carp brain cross-sections; L Immunohistochemistry of Gαq protein in grass carp pituitary. Red arrows indicate areas of high expression. OB, olfactory bulb; Te, telencephalon; OT, optic tectum; Hy, hypothalamus; Ce, cerebellum; MO, medulla oblongata; ML, molecular layer; GL, granular layer; TL, longitudinal restraint; Pit, pituitary; NH, neurohypophysis; RPD, rostral pars distalis; PPD, proximal pars distalis; PI, pars intermedia

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The distribution of Gαq and luteinizing hormone receptor (LHR) in the ovary and testis of the grass carp was analyzed using immunofluorescence co-localization. The results showed strong signals for both Gαq and LHR in the testis and ovary (Fig. 6 and S5). However, contrary to the widely accepted belief that LHR is localized to the cell membrane as a GPCR, our findings revealed that LHR is primarily located in the nucleus of grass carp oocytes, with some signals also present in follicular cells (Fig. 6 and S5). On the other hand, Gαq protein was mainly found in the cytoplasm of oocytes, with no signal detected in follicular cells. In the testis, Gαq protein and LHR were co-localized in the cytoplasm (Fig. 6 and S5).

Fig. 6
figure 6

Colocalization of Gαq and LHR fluorescence in the ovary and testis of grass carp. A Ovary; B testis. Blue, green, and purple indicate DAPI, Gαq, and LHR staining, respectively. The images in the bottom left (A) or bottom right (B) corners are enlarged images

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Regulation of Cilhr and Cipgr expression by Cignaq (Gαq)

The expression of grass carp reGαq is showed in Fig. S6. GCO cells were incubated with grass carp reGαq (30ug/well, six-well plate) and the results showed that reGαq did not significantly promote Cilhr expression in GCO cells (P > 0.05; Fig. 7A). However, it did significantly promote Cipgr expression (P < 0.05; Fig. 7C). When reGαq was co-incubated with hCG, there was a significant increase in the expression of both Cilhr and Cipgr compared to the control (P < 0.05; Fig. 7B and D).

Fig. 7
figure 7

Regulation of Cilhr and Cipgr expression by grass carp recombinant Gαq protein. A Cilhr expression after incubating GCO with Gαq; B Cilhr expression after incubating GCO with Gαq and hCG; C Cipgr expression after incubating GCO with Gαq; D Cipgr expression after incubating GCO with Gαq and hCG. * P < 0.05; *** P < 0.001

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To further study the regulation of Cignaq on Cilhr and Cipgr, siRNA was used to silence Cignaq expression. The results showed that siRNA successfully interfered with Cignaq expression (P < 0.05; Fig. 8A and B). After siRNA silencing Cignaq expression, the expressions of Cilhr and Cipgr were relatively decreased compared to the control group, although the decreases were not significant (P > 0.05; Fig. 8). When GCO cells were stimulated with hCG after siRNA silencing Cignaq expression, the expression of Cipgr in the interference group was significantly lower than that in the control (P < 0.05; Fig. 8C). In contrast, the expression of Cilhr in the interference group was relatively higher than that in the control group, although there was no significant difference (P > 0.05; Fig. 8D).

Fig. 8
figure 8

Regulation of Cipgr and Cilhr expression by siRNA silencing of Cignaq. A Cignaq expression after siRNA silencing; B Cignaq expression after hCG-stimulated siRNA silencing; C Cipgr expression after siRNA silencing of Cignaq; D Cilhr expression after siRNA silencing of Cignaq. * P < 0.05; ** P < 0.01

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Discussion

In numerous GPCR signaling pathways, the Gα family acts as a molecular switch, transitioning between inactive and active states by binding to GDP [20]. This family is typically divided into four functional subfamilies [25], each triggering a distinct cascade of events [19]. Interestingly, this study found a larger number of Gα genes in the grass carp genome (27) compared to the human genome [20].

Gene duplication plays a crucial role in expanding gene families by creating gene clusters through tandem repeats and segmental duplication. This process results in the formation of homologous genes, ultimately increasing the total number of genes [49]. The grass carp genome has a large number of Gα genes, which may be attributed to fish-specific duplication events that aid in the species’ adaptation to diverse aquatic environments [58]. Our findings indicate that the grass carp genome contains 27 Gα genes, with eight being the result of tandem duplication and three from segmental duplication. Furthermore, our analysis suggests that purifying selection was the primary driving force behind the evolution of the Gα family. Additionally, we identified a unique gene, Cignav1, which has no orthologous genes in mammals and may play a role in regulating cell osmolality and taste bud differentiation [59].

The proportion of Gαi/o subfamily members was found to be higher in both mice and humans [45]. Our dendrogram tree analysis revealed that the Gα family of grass carp can be divided into four distinct subfamilies, with the Gαi/o subfamily containing the largest proportion (44.4%) of Gα genes. During evolution, the gene structure of Gα family members has undergone significant changes, resulting in a variation in the number of exons, ranging from 4 to 12. Specifically, the Gα12/13, Gαq/11, Gαi/o, and Gαs subfamilies contained 4, 7, 8–9, and 11–12 exons, respectively. This suggests that the number of exons is a characteristic feature of each subfamily [60, 61]. Our analysis also revealed that the Gα family members of grass carp share high conservation in terms of domain and motif sequences. This indicates that the Gα family plays a fundamental role in binding to GDP or GTP, βγ-complexes, and receptors [25, 33].

Each Gα subunit typically couples with multiple GPCRs, resulting in a unique cellular response [62]. The C-terminal tail of the Gα subunit plays a significant role in the interaction with GPCRs, accounting for approximately 70% of the interacting surface and contributing to the selectivity of GPCRs [22, 63, 64]. In the HPG axis transcriptome of grass carp, we observed distinct expression patterns for 15 Gα family members, with specific tissue expression characteristics. However, the expression pattern was not solely determined by the Gα subfamily, as genes from different subfamilies also exhibited similar expression patterns.

Serotonin plays a crucial role in regulating the reproductive function of fish through various mechanisms [65]. It binds to 5-HTR2, which in turn regulates excitatory neurotransmission through Gαq/11 and stimulates GnRH release in Pagrus major [66]. Additionally, neurokinin B activates the PKC/Ca2+ signaling pathway through Gαq/11 or the PKA/cAMP signal transduction pathway through Gαs to regulate reproduction [67]. Our findings demonstrate that Gαq can induce the expression of Cipgr. Furthermore, under the stimulation of hCG, the effect of Gαq on Cipgr expression was more pronounced, and Gαq also promoted the expression of Cilhr. To further investigate the role of Gαq, we utilized siRNA to interfere with the expression of Cignaq and observed a decrease in Cipgr expression even under hCG stimulation, while Cilhr expression increased. These results suggest that Gαq functions similarly in grass carp as it does in mammals, mediating the high expression of Cipgr induced by LH and triggering various physiological activities, ultimately promoting ovulation.

In this study, we used grass carp with different ages to investigate the role of the gnaq at various developmental stages. Previous studies have shown that the KISS1/GPR54 system regulates the onset of puberty, and mutations in the GPR54 gene have been linked to sexual maturation disorders [68,69,70]. GPR54 couples with gnaq/gna11 proteins, activating PLCβ, which then triggers downstream signaling molecules to hydrolyze PIP2 into DAG and IP3. The increase in IP3 stimulates the endoplasmic reticulum (ER), leading to the mobilization of intracellular Ca2+. This rise in Ca2+ levels activates calcium-dependent signaling pathways in GnRH neurons, thereby regulating GnRH secretion [38, 39]. We specifically chose 150-day-old grass carp for this study because they are at a critical stage of gonadal development. Grass carp have a long sexual maturation cycle, typically lasting 4–6 years, which may vary depending on climatic conditions. One-year-old grass carp are more readily available. Therefore, we chose to use them for the gnaq gene tissue expression profile. Additionally, gnaq has been reported to affect estrus and reproductive processes in animals [71]. In fact, previous research has shown that mice with a gnaq/gna11 knockout experience reproductive defects, with female mice unable to spontaneously ovulate [40]. Therefore, to further investigate the role of gnaq in fish ovulation, we chose to use four-year-old grass carp. The gnaq plays a crucial role in cellular signaling and has significant implications for fish reproduction. However, it is important to note that our study only uncovers a small part of the role of gnaq in reproduction.

Conclusions

Twenty-seven Gα genes with 46 isoforms had been identified in the grass carp genome. These genes had been divided into four subfamilies: Gαs (Cignas, Cignal, and Cignal2), Gαi/o (Cignai, Cignai1, Cignaia, Cignai2a, Cignai2b, Cignat1, Cignat2, Cignaoa, Cignaoa-like, Cignaob, Cignaz, and Cignav1), Gαq/11 (Cignaq, Cigna11, Cigna11-like, Cigna11a, Cigna11b, Cigna14, Cigna14-like, and Cigna15.1), and Gα12/13 (Cigna12, Cigna12a, Cigna13a, and Cigna13b). The Cignaq sequence was highly conserved and highly expressed in the HPG axis. It functions were similarly to mammalian Gαq and plays a crucial role in reproductive regulation. However, the specific regulatory mechanisms upstream and downstream of the HPG axis require further exploration. These results provided preliminary evidence for understanding the role of Gαq in the reproductive regulation in teleosts.

Availability of data and materials

The transcriptome sequencing datasets generated and analyzed during the current study are available in the Sequence Read Archive repository, accession number PRJNA1146714. All other relevant data are available from the authors upon request and the corresponding author will be responsible for replying to the request.

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Acknowledgements

We would like thank Guangdong Meilikang Bio-Science Ltd., China for their assistance in data analysis.

Author statement

The manuscript has been read and approved by all authors and there are no other persons who satisfied the criteria for authorship but are not listed.

Funding

This study was supported by the Natural Science Foundation of Hunan Province (Grant No. 2022JJ30289).

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Author notes

  1. Chong Wang and Shuting Xiong the authors contributed equally to this work.

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  1. Fisheries College, Hunan Agricultural University, Changsha, 410128, China

    Chong Wang, Shuting Xiong, Shitao Hu, Le Yang, Yuhong Huang, Haitai Chen, Baohong Xu, Tiaoyi Xiao & Qiaolin Liu

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CW, QLL, STX, BHX and TYX: conceived and designed the experiments. CW, SH, LY, YHH, and HTC: performed the experiments. CW: analyzed the data and wrote the original draft. QLL: writing-review, editing the manuscript, and formal analysis. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Tiaoyi Xiao or Qiaolin Liu.

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The fish used in this study were obtained with the informed consent of the owners for research purposes. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Hunan Agricultural University.

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Not applicable.

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The authors declare no competing interests.

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Wang, C., Xiong, S., Hu, S. et al. Genome-wide identification of Gα family in grass carp (Ctenopharyngodon idella) and reproductive regulation functional characteristics of Cignaq. BMC Genomics 25, 800 (2024). https://doi.org/10.1186/s12864-024-10717-0

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  • DOI: https://doi.org/10.1186/s12864-024-10717-0

Keywords

BMC Genomics

ISSN: 1471-2164

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