Cloning of estrogen receptors (ERs)

To clone “a partial cDNA fragment of ERα, ERβ1, and ERβ2, primers, as shown in Table 1, were designed”. Primers were designed for amplification of hybrid grouper ERα, ERβ1, and ERβ2 gene coding sequences using the existing full-length sequences of Epinephelus coioides-estrogen receptor alpha -(GU721076.1); Acanthopagrus schlegelii-estrogen receptor alpha-(AY074780.1); Sparus aurata-estrogen receptor alpha-(AF136979.2) and Acipenser schrenckii-estrogen receptor alpha1-(AB435631.1) ; Epinephelus coioides estrogen receptor beta 1-(GU721077.1); Sparus aurata-receptor type beta1-(AF136980.1); Acanthopagrus schlegelii –estrogen  receptor  beta1-(AY074779.1), and  Acipenser schrenckii-estrogen receptor beta1-(AB435633.1); Epinephelus coioides estrogen receptor beta2- (GU721078.1); Acanthopagrus schlegelii estrogen receptor beta 2-( EU346949.1) and Micropterus salmoides estrogen receptor beta2-( AY211021.1).

Primer 5 software was used to design primers (Table 1). cDNA synthesis was performed using the TRANSgen First-strand cDNA synthesis kit with a total volume of 20 μl of reaction mixture following the manufacturer’s guidelines. The partial cDNA fragments of ERs were “amplified from the first-strand cDNA from” a mix of the liver, heart, and brain tissues. The PCR reaction was performed in 20 μl volumes of the reaction mixture. The amplification was performed following reaction conditions, “94°C for 5 min, followed  by  35  cycles  for 30 s at 94°C, for 30 s at 58°C, for 35 s at 72°C and 10 min at 72°C”. The separation of PCR products was done by using electrophoresis, and the “DNA Bands were recycled and purified by the use of the SMART RACE cDNA purification Kit (Clontech, Palo Alto, CA)”. The “purified DNA portions were then subcloned into the pMD18-T vector (Takara, Japan) and converted into competent Escherichia coli DH5a cells”. A maximum of “three different positive clones each were selected and send to Sangon Biological engineering (Guangzhou) LTD”. for sequencing (Amenyogbe et al., 2019). To “clone 3Ò† and 5Ò† untranslated region (UTR) end of ERα, ERβ1 and ERβ2, two sense primers were designed depending on the partial cDNA sequence of ERα, ERβ1 and ERβ2 as shown in Table 1 using the SMART RACE cDNA Synthesis Kit (Clontech, Palo Alto, CA) according to the manufacturer’s guidelines”. A “clear fragment was purified, cloned, and sequenced using the same procedure as described earlier”.

3′ RACE was done in “both first and second amplification” using Amenyogbe et al. (2019) method. Additionally, “5′ RACE was carried out using the same procedures and methodologies that made use of reverse primers (Table 1) in place of sense primers”. “PCR amplification was done in a 15-µl volume of the reaction mixture” following Amenyogbe et al. (2019) method.

Sequence analysis

The “sequences of partial, 3′ and 5′ UTR were assembled to form the full-length cDNA of the target gene by the use of LaserGene software” (https://dnaman.software.informer.com). The “Gene translation, predictions of the amino acid sequence” was done using EXPASY web tools (http://expasy.org/tools). The conserved domains search was done using Marchler-Bauer et al. (2011) to ascertain conserved domain sequences among designated ER superfamily genes. To establish genetics relations, phylogenetic analysis was carried out, and a consensus tree was built using MEGA 6.0 software 6 (Tamura et al., 2013). We examined the physical and chemical properties of the protein sequences using the PROTEAN program (version 5.07; DNASTAR Inc.: Madison, WI, USA, 2003.) to ascertain their chemical compositions. COFACTOR server (Zhang et al., 2017) method was used for gene ontology GO terms predictions.

Tissue mRNA expression of ERs by qRT-PCR

The “mRNA levels of ERα, ERβ1, and ERβ2 in tissues were resolute by Real-time qRT-PCR” (Amenyogbe et al., 2019) in the liver, muscle, brain, stomach, spleen, intestine, gill, head kidney, kidney and heart using Transtar “Tip Green qPCR Supermix (TransGen Biotech, China)” the manufacturer´s instructions were followed, a Bio-Rad Connect (“Roche Light Cycler®96 SW1.1” Real-time Detection) in 10 ul reaction. We used β-actin as a control gene. The primers for ERα, ERβ1, and ERβ2 and β-actin were designed using primer5 designers (Table 1). The total mixture for the reaction for quantitative Real Time-PCR consisting of 5 ul Transtar “Tip Green qPCR Supermix (TransGen Biotech, China), and 0.4 ul of each sense and antisense primer, 3.6 ul of H₂O and 0.6 ul of cDNA”. A melting curve was performed to detect the specificity (Fu et al., 2014). The “reaction conditions of for the qRT-PCR were as follows: 30 s at 95°C; and was amplified for 40 cycles for 15 s at 95°C, annealing for 15 s at 59°C and extension for 15 s at 72°C”. Each sample “verified in triplicate”. The 2−ΔΔCt methods (Livak, and Schmittgen, 2001) was used to calculate the results.

Statistical analysis

The  data   in  this  study  were  articulated  as  a  means±SEM. The significant differences in the data between ERs are presented using one-way ANOVA followed by Duncan’s posthoc test and a probability level less than 0.05 (P < 0.05) was used to indicate significance. Also, the Independent samples T-test was also used, and the significance level was set at P < 0.05. All statistical analysis were performed using SPSS 16.0 (SPSS, Chicago, IL, USA) (Cui et al., 2017).

Cloning and characterization of hybrid grouper ESRs

By the use of standard PCR procedures, the partial DNA fragments were augmented from Hybrid grouper (Epinephelus fuscoguttatus â™€× Epinephelus polyphekadion ♂) liver, heart, and brain RNA. The DNA fragment acquired, and sequence examination exhibited that the fragments had a resemblance to ERα, ERβ1, and ERβ2. The RACE technique was used “to clone full-length” of hybrid grouper ERα, ERβ1 and ERβ2 cDNAs with the following (GenBank accession no. MK575468, MK544841, and MK570511 for ERα, ERβ1, and ERβ2 respectively. The sequence analysis of cDNA for ERα show 621 amino acids and considered molecular mass 61.04 kDa and ERβ1 comprises 546 amino acids and a determined molecular mass 60.47 kDa, and ERβ2 show 244 amino acids and a determined molecular mass 27.49 kDa. The hybrid grouper ERs “sequence can be grouped into five domains based on its sequence identity to other species’ ERs” (Figure 2) as suggested by Katsu et al. (2010c). Amino acid sequences of hybrid grouper hgERα shared the identity of 42.3 and 40.3% with hgERβ1 and hgERβ2 respectively while hgERβ1 show the identity of 56.8% with hgERβ2. Hybrid grouper sequences of ERs equated with different species (Homo sapiens, Mus musculus, Epinephelus coioides, Dicentrarchus labrax, Oreochromis aureus, Sebastes schlegelii, Acanthopagrus latus and Sparus aurata), Hybrid grouper ERα shared 81.9-98.8, and 57.4-958% identities in the C, and E domains, respectively (Figure 1A). In contrast, Hybrid grouper ERβ1 shared 63.3-97.5, and 66.1-98.7% identities in the C, and E domains, respectively (Figure 1B) and hybrid grouper. The clone protein sequence of ERβ2 shared 22.9-75.2% identities in the E domains. Therefore, “C domain or DNA-binding domain and E domain or the ligand-binding domains” were conserved in all vertebrate ERs considered. The general identities of hybrid grouper ERα with ERα in (Homo sapiens, Mus musculus, E. coioides, D. labrax, and S. schlegelii) were 46.1, 46.6, 89.1, 88.3, and 90.2%, respectively, the overall identities of hybrid grouper ERβ1 with ERβ1 from the (Homo sapiens, Mus musculus, E. coioides, O. aureus, and S. schlegelii,) were 48.2, 48.3, 91.5, 79.0, and 85.7%, respectively. The overall identities of hybrid grouper ERβ2 with ERβ2 from the (Homo sapiens, Mus musculus, E. coioides, A. latus, and S. aurata) species were 44.9, 44.4, 74.5, 69.1 and 70.0% respectively. The phylogenetic  analysis  showed  that  each  of  these ERs belongs to ER superfamily (Figure 3).

The sequence alignments of ERs of different vertebrates including Hybrid grouper are demonstrated indicating the various domains. The Genbank accession numbers of ER sequences used are as same as ones used in the phylogenetic analysis. The MAPK, “P, and D-boxes from  DBD”,  cAMP,  PKC,  AF-2  are  indicated  by Elbow Double-Arrow connector. The down arrow indicates the eight “cysteine residues of the zinc-finger motif”. “A/B, B/C, C/D, D/E, and E/F” domains are marked with Double Arrows.

The phylogenetic examination was done using “Clustral W”. The different ER sequences obtained from the Genebank  database.  Genebank  accession  numbers of the sequences are: hybrid grouper ERα, MK575468; E. coioides ERα, ADK90033; D. labrax ERα, CAD43599; S. schlegelii ERα, ACN39246; Mus musculus-estrogen receptor isoform 1, NP_031982; Homo sapiens-estrogen receptor isoform 1, NP_000116; hybrid grouper ERβ1, MK544841; E. coioides ERβ1, ADK90034; O. aureus ERβ1, ACF75102; S. schlegelii ERβ1, ACN38898; Mus musculus-estrogen receptor beta isoform 2, NP_034287; Homo sapiens-estrogen receptor beta isoform 1,NP_001428 XP_495993; Hybrid grouper ERβ2, MK570511; E. coioides ERβ2, ADK90035; A. latus ERβ2, AEX68679; S. aurata ERβ2, CAE30471; Mus musculus, NP_034287 and Homo sapiens, NP_001258805. Where EFERbeta1- hybrid grouper ERβ1, SsERbeta1- S. schlegelii ERβ1, OgERbeta1- E. coioides ERβ1, mOUSEERb1- Mus musculus, ToERbeta1- O. aureus, SaERbeta2- S. aurata ERβ2, EcERa- E. coioides ERα, mouERb2- Mus musculus, DIERa- D. labrax ERα, HgERa-hybrid grouper ERα, SCERa- S. schlegelii ERα, AlERbeta2- A. latus ERβ2, humanERb1- Homo sapiens, HuERb2- Homo sapiens, HgERbeta2-hybrid grouper ERβ2, MmER1- Mus musculus and HsER1- Homo sapiens.

Gene expression

While at a different level, hybrid grouper estrogen receptors (hgERs) were expressed in all tissues samples that were studied. The hgERα expression level was found to be highest in the heart among the ten tissues examined, followed by the muscle. The hgERβ1 was noticeably paramount also in the heart, followed by the liver. The hgERβ2 expressed most profoundly in the liver, followed by the stomach. Generally, the highest mRNA expression levels of hgERα, hgERβ1 and hgERβ2 were all found in the heart. There was a substantial difference between the expression in the heart and liver and other samples as shown in Figure 4a, b, and c. All hybrid grouper ERs studied were expressed in all tissues samples examined, although at different levels. Interestingly, hgERα expressed relatively higher in all tissues as compared to the hgERβ1 and hgERβ2.

Estrogen receptors play very important traditional roles in the development of the reproductive system in vertebrates mostly in gonads and testis. Understanding and pinpointing the dissemination of ERs in hybrid grouper will help in understanding the possible other roles of estrogen in hybrid grouper development. Consequently, to our knowledge and understanding, there has been no study of the expression or the role of the ER in juvenile hybrid grouper a fish that has not developed gonads or testis yet. Estrogen is known traditionally to have a multiplicity of physiological functions and is tangled in regulating vertebrate metabolism, reproduction, cell proliferation, differentiation and inflammation through cellular machinery (estrogen receptors) required to warrant that estrogen executes these functions. Reproduction “activities in vertebrates, such as gonadal differentiation, maturation of the female reproductive tract, and procreative behaviors” are all associated with estrogen (Moore et al., 2005; Iguchi et al., 2001; McLachlan, 2001). In vertebrates, “estrogens seem to persuade both genomic and non-genomic cellular actions through the nuclear and perhaps G-coupled membrane receptors” (Moore et al., 2005; Iguchi et al., 2001; McLachlan, 2001). In 1990, the rainbow trout ER full-length sequence was reported in fish (Katsu et al., 2010c; Bjornstrom and Sjoberg, 2005). Ever since several other sequences have been recounted for teleost fishes, and three different types of ERs have been sequestered to date in a teleost (Katsu et al., 2010c; Hawkins et al., 2000).

In this study, full-length cDNA “sequences of distinctive ERα, ERβ1, and ERβ2” were cloned (Hu et al., 2018) from the juvenile hybrid grouper and characterized using PCR and the position of expressed ERs in the various tissues were examined. Hence, the prospective role of ERs in juvenile hybrid grouper development can be implicit. Upon aligning the amino acid sequence of the gene and sequences from different fish species, it has been found that the cDNA sequence of hybrid grouper estrogen receptors and their deduced amino acid sequence replicated a high degree of homology with the ERs homologs recognized from other animals and also the ERα and ERβ1 of  juvenile hybrid  grouper  consist  of well-known A/B, C, D, E and F molecular domains (Figure 2). This is an indication that this newly isolated cDNA encoded the hybrid grouper ERs protein.

Compared to other teleost fish, the A/B domain of the juvenile hybrid grouper ERα, and ERβ1, the C and E domains  were  less  conserved (Ding et al., 2016). It was also noted that the “phosphorylation sites known by mitogen-activated protein kinase (MAPK) were existent in the A/B domain of ERα and ERβ1”, an indication that ERα and ERβ1 may influence the activation of MAPK pathway (Kato et al., 1995; Ding et al., 2016). It  has  also been noted that the AF-2 Activation domain (DLLLEML) occurred between amino acid residues 535 and 549 of the LBD domain, indicating that transcriptional activity was dependent on ligand binding (Ding et al., 2016; Kumar et al., 1987).

The hybrid grouper ERs genes shared high ranks of protein distinctiveness between the “DNA-binding domains” and contained the conserved motifs and elements believed to be essential for specific nuclear localization and command for target genes (Cui et al., 2017; Hall et al., 2002). There was also the reasonable uniqueness in the area called E/F domains or ligand-binding domains of the estrogen receptors which may be accountable “in part for the ligand-specificity and the different” answers to estrogen (Cui et al., 2017; Danielian et al., 1992; Kumar et al., 1987). The “ligand-binding domain”, the AF-2 which is the “estrogen-dependent activation domains” were also observed to be conserved, indicating the similarity with other fishes, including Scatophagus argus, E. coioides, and S. schlegelii (Cui et al., 2017; Chen et al., 2011; Kim et al., 2003). Accepted functional sites of the protein for hgERs exhibited consistency with ERs. It is observed that the following domains “cysteine residues for two zinc fingers, P-box, D-box, and a cAMP site in the DBD domain, an AF-2 site, and a PKC phosphorylation site in the LBD domain” are conserved in hgERs (Figure 2). According to Mu et al. (2013), P- and D-boxes are shown to be crucial for DNA-binding. The importance of “PKC sites in all ERs” publicized by Härd and Gustafsson (1993) in which the initiation of PKC noticeably improves “ER-mediated transcriptional activation in a ligand-dependent manner” (Fu et al., 2008). The recognized A/B domain which contains the MAPK phosphorylation site was also observed to be conserved in the ERα subtype in sequence and position, as noted by others (Kato et al., 1995; Cho and Katzenellenbogen, 1993). Socorro et al. (2000) reported that the MAPK pathway could influence the “ligand-independent transcriptional activity of ER in both mammalian ERα and ERβ” (Fu et al., 2008).

While increasing literature exists on a phylogeny for numerous vertebrate steroid receptors (Howarth et al., 2008; Bury and Strum, 2007; Pakdel et al., 1990), scarce literature is available on hybrid species. Our study enhances essential material in this catalog. The analysis of sequences showed that two ERs protein sequences made up of the unique domain structures for NR superfamily while the hgERβ2 demonstrated the difference (Hu et al., 2006) slightly. A cautious examination of the phylogenetic tree discovered that this is in agreement with the case for ERs. At least “three sub-clusters of ERs were found in juvenile hybrid grouper even though maximally two ERs subtypes were isolated in many species” (Wang et al., 2005). The two ER-b subforms were reported in species of teleosts, such as the “Atlantic croaker, the Nile tilapia, and fugu, though not distributed in the same two sub-clusters as did the two ER-bs from zebrafish and goldfish” (Wang et al., 2005). An indication that “at least one of the two ER-b subtypes in the tilapia, Atlantic croaker, and fugu, tilapia-fugu ER-b2 clade has a diverse source from those of the zebrafish and goldfish, zebrafish ER-b1 clade” (Wang  et  al.,  2005; Robinson-Rechavi et al., 2001b). It is possible that two consecutive lineage-specific replications might have transpired independently. Together they “took place after the divergence of teleosts”. It is possible “the former took place only in zebrafish lineage, and the latter” transpired in the other teleosts deprived of the zebrafish lineage. The findings of the present study backed the proposition that most replicas of “fish genes arose more recently than the divergence of major fish groups” (Wang et al., 2005; Robinson-Rechavi et al., 2001b).

Examination of the tissue distribution of ERα, ERβ1, and ERβ2 offers comprehension into the prospective for targeting specific tissues. In the present research, we examined the expression configuration of ERs that is ERα, ERβ1 and ERβ2 mRNA in diverse tissues of juvenile hybrid grouper. The study revealed that ERs was expressed in all tissues of juvenile hybrid grouper examined. In goldfish, gilthead seabream and gilthead seabream the estrogen receptors were found to express mainly in gonads, but the overall expression in heart, liver, stomach, muscle intestine and head kidney showed largely corresponding expression patterns in hybrid grouper (Kato et al., 1995), the profiles of hgERs expression are similar to previously reported in tissue samples that were studied for all ERs in sea bass, with similar echelons among tissues (Lannigan, 2003) indicating possible similar function in hybrid grouper. The analysis of tissue expression in various tissue samples discovered all the three estrogen receptors expressed widely in hybrid grouper tissues which are in agreement with results from other studies (Cui et al., 2017; Cheng et al., 2015; Chen et al., 2011; Filby and Tyler, 2005; Halm et al., 2004; Kim et al., 2003) with the highest expression level in the heart contrary to study in goldfish which reported that ERα mRNA expression level was highest in the pituitary gland (Choi and Habibi, 2003). The expression of three estrogen receptors at differential expression configurations in tissues indicates that they might have diverse physiological roles. The hybrid grouper ERs gene was found to be highest in the heart but significantly lower in the kidney. Studies on S. aurata reported that ERα was expressed mainly in the liver and pituitary gland (Ding et al., 2016; Pinto et al., 2006), in partial agreement with the present study, whilst Oreochromis mykiss ERα and ERβ1 was found to expressed highest in the lever (Nagler et al., 2007). Altogether, the three estrogen receptors expressed highly in the heart, muscle, and liver, which suggests all of them, may be involved in growth and reproduction regulation in hybrid grouper (Epinephelus fuscoguttatus â™€× Epinephelus polyphekadion ♂). The ERs expression levels were comparatively high in the heart of ERα, ERβ1, and ERβ2. Even though ERs expressed in the liver, and the manifestation levels were low, this is a contradiction to other studies. This finding was contrary to the expression patterns of ERs in goldfish, S. aurata, and O. mykiss.  The  reason  for these occurrences is not readily known and further study is necessary to elucidate the implications.

This study established the actuality of three estrogen receptors in juvenile hybrid grouper and demonstrated that ER-alpha, ER-beta1 and ER-beta2 are expressed throughout all tissues which implies that estrogen through these receptors may be responsible for the regulations of physiological and pathological functions in Hybrid grouper. The copious expression of hybrid grouper ERs advocates a broad expression pattern as in mammalian ERs. These results put forward that steroid hormone estrogen receptors might be playing a significant part in the controlling of social behavior complexity, plasticity behavior, and the assessment of a gratifying inducement in Hybrid grouper. Based on this study, further study is necessary to elucidate the effect of ERs in developmental stages.

There is also need for research of the spatial configurations of ER-transcript expression in adult hybrid grouper tissues and also further dichotomization of the part estrogen receptors might be playing in regulating the incredible malleability of social behavior within hybrid grouper.

This exertion is supported by the China Agriculture research system (CARS-47) and Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (ZJW-2019-06).

The authors have not declared any conflict of interests.