Unique oestrogen receptor ligand-binding domain sequence of native parrots: a possible link between phytoestrogens and breeding successCatherine E. J. Davis A , Adrian H. Bibby A , Kevin M. Buckley A , Kenneth P. McNatty A and Janet L. Pitman A B
A School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand.
B Corresponding author. Email: email@example.com
Reproduction, Fertility and Development - https://doi.org/10.1071/RD17045
Submitted: 7 February 2017 Accepted: 1 June 2017 Published online: 11 July 2017
The New Zealand (NZ) native parrots kākāpō, kākā and kea are classified as critically endangered, endangered and vulnerable respectively. Successful reproduction of kākāpō and kākā is linked to years of high levels of fruiting in native flora (mast years). To assess a possible hormonal link between native plants and reproductive success in these parrots in mast years, we examined the ligand-binding domains (LBD) of the progesterone receptor (PR), androgen receptor (AR), estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) in NZ native (kākāpō, kākā, kea and kākāriki) and non-native (Australian cockatiel) parrots and compared them with those in the chicken. The amino acid sequences for PR, AR, ESR1 and ESR2 shared >90% homology among the NZ parrots, the cockatiel and, in most cases, the chicken. The exception was for the ESR1 LBD, which contained an extra eight amino acids at the C-terminal in all the parrots compared with the chicken and with published sequences of non-parrot species. These results support the notion that the ESR1 LBD of parrots responds differently to putative oestrogenic compounds in native trees in NZ during times of intermittent masting. In turn, this may provide important information for generating parrot-specific bioassays and linkages to steroidogenic activity in native plants.
Additional keywords: kākāpō, masting, steroid receptor.
A large number of New Zealand (NZ) avian species, including those belonging to the Order Psittaciformes, have declining populations linked to habitat loss and an increasing number of predators. These species include the critically endangered kākāpō (Strigops habroptilus), the endangered kākā (Nestor meridionalis), the vulnerable kea (Nestor notabilis), and kākāriki (Cyanoramphus spp.). With around 150 surviving kākāpō, it is now listed among the top 10 most endangered species worldwide (Department of Conservation National Office 2017).
The reproductive strategies between the NZ parrot species are different. Kākāpō have a low reproductive rate, and successful breeding is linked to the years in which masting (high levels of fruiting) occurs in native plant species such as rimu (Dacrydium cupressinum; Powlesland et al. 1992; Elliott et al. 2001; Cockrem 2006). In contrast, kākā are episodic breeders and are capable of breeding more than once in a season, whereas kea tend to breed annually. Kākāriki can breed multiple times in a year, although this is reliant on sufficient food being available (Elliott et al. 1996; Elliott and Kemp 1999; Higgins 1999; Greene et al. 2004).
Although kākāpō and kākā are known to breed successfully during masting years (Powlesland et al. 1992; Wilson et al. 1998; Greene et al. 2004; Cockrem 2006), high fruit abundance is sporadic and often absent in consecutive years, coincident with poor reproductive success, especially in kākāpō (Schauber et al. 2002; Cockrem 2006). Recent attention has focused on potential linkages between the likely stimulants in plants that may augment or activate reproductive activity in kākāpō and kākā (Elliott et al. 2001; Cockrem 2006). One possibility relates to the steroidogenic activities in the fruits of certain native plants that are browsed by these parrots (Fidler et al. 2000, 2008).
Numerous studies have investigated steroid receptors in different species but there is an absence of information in parrots. In mammals, steroid receptors for progesterone (i.e. progesterone receptor (PR)), androgen (i.e. androgen receptor (AR)) and/or oestrogen (i.e. estrogen receptors 1 and 2 (ESR1 and ESR2 respectively)) are critical mediators of steroid actions in the reproductive system (Dupont et al. 2000; Conneely et al. 2001, 2003; Drummond et al. 2002). Variants in the steroid receptors as a result of mutations or splicing have been shown to affect ligand binding and/or transcriptional activity (Bai and Weigel 1996; Ong et al. 2002; Deroo and Korach 2006; Zhou et al. 2011). Thus, differences between avian species in the ligand-binding domain (LBD) of specific steroid receptors may have profound effects with regard to their responses to plant hormones.
Steroid receptors possess a modular structure that includes an N-terminal regulatory domain, a DNA-binding domain (DBD), a hinge domain, an LBD and a C-terminal domain. Both the DBD and LBD exhibit high levels of conservation (Deroo and Korach 2006). Because the LBDs provide information about likely interactions with plant hormones, these were considered to be the regions of focus for the present study.
In the present study, we elucidated and compared the LBD of the steroid receptors PR, AR, ESR1 and ESR2 in kākāpō, kākā, kea, kākāriki, the Australian cockatiel (Nymphicus hollandicus) and the Japanese quail (Coturnix japonica) using sequences from the chicken as the reference source. The amino acid sequence of kākāpō ESR1 was then used for in silico modelling to compare the most favourable binding position of oestrogenic ligands in the putative three-dimensional structure with that of the fully characterised human ESR1.
Materials and methods
Approval to collect and store biological samples from native bird species were obtained from the NZ Department of Conservation and the NZ Environmental Risk Management Authority (ERMA). Steroid receptor variants have been reported in a broad range of tissues (Leung et al. 2006). This is of value in identifying potential tissue-specific variants of steroid receptors when different tissues are available for study (Sotoca et al. 2012). Thus, for the NZ parrots, Japanese quail, cockatiel, and chicken, a minimum of three different tissue types from either the brain, heart, kidney, liver, muscle, ovary or testis were sampled, and each sample type was collected from at least three different individuals.
Polymerase chain reaction
Total RNA was isolated from kākāpō, kākā, kea, kākāriki, cockatiel, Japanese quail and chicken tissues using TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. The isolated RNA was then treated with DNase I (RiboPure-Blood Kit; Ambion) and quantified using an Agilent RNA 6000 Nano Assay Kit (Agilent Technologies). Thereafter, cDNA was synthesised from 5 µg RNA using Superscript III First-Strand Synthesis Supermix Kit (Life Technologies) according to the manufacturer’s instructions. The quality of the cDNA was assessed by performing polymerase chain reaction (PCR) with primers designed for the β-actin (ACTB) gene (data not shown).
Primers were designed for the four steroid receptors using GenBank sequences for chicken (AR, NM_001040090; ESR1, NM_205183; ESR2, NM_204794; PGR, NM_205262) and manufactured by Life Technologies (Table 1). Each PCR reaction (total volume 50 µL) consisted of cDNA template (equivalent of 1 µg RNA), 200 µM of each dNTP (Life Technologies), 0.2 µM of forward and reverse primers, 5 µL of 10× HotMaster Taq Buffer (5 PRIME) and 1.5–2.5 U HotMaster Taq DNA Polymerase (5 PRIME). The following cycling conditions were used: an initial denaturation at 95°C for 3 min, followed by 35–40 cycles of 95°C for 20 s, 58°C for 25 s and 72°C for 50 s, with a final extension at 72°C for 7 min.
DNA sequencing and protein translation
The resulting amplicons were visualised on an agarose gel with SYBR green. The appropriate bands were extracted from the gel using a QIAquick Gel Extraction Kit (Qiagen) following the manufacturer’s instructions. The eluted DNA was then ligated into 50 ng pGEM-T Easy plasmid vector using 3 U T4 DNA Ligase (Promega). The vector containing the DNA insert was transformed into DH5α cells (Life Technologies) and incubated onto plates containing LB agar (Becton Dickinson) and 50 µg/mL ampicillin (Sigma-Aldrich) and 2% X-gal (VWR International) for 18 h at 37°C. Individual colonies were selected and grown in luria broth containing 50 µg mL-1 ampicillin at 37°C for 18 h. DNA was purified using a QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s instructions. Sequencing of the amplified DNA was performed by Waikato DNA Sequencing Facility (University of Waikato). The resulting sequences were translated and aligned using Geneious bioinformatics software (www.geneious.com, ver. 5.5.8).
In silico protein modelling
To stimulate protein–ligand interactions and identify low energy complexes, in silico modelling using RosettaLigand software (ver. 3.5; downloaded from http://www.rosettacommons.org) was performed for kākāpō ESR1 LBD. The docking studies were performed on Victoria University of Wellington’s Science Faculty High Performance Computing Facility, using computer nodes comprising two 12-core Advanced Micro Devices processors running at 2.2 GHz. The crystal structure of human ESR1 (1A52) was obtained from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/home/home.do, accessed 2012), modified to contain the residues of kākāpō ESR1 and the structure was then minimised. The parameters for docking runs included 50 translation iterations and 1000 rotation iterations. After each set of iterations, the file was optimised 100 times through a high-resolution docking protocol. The total number of runs for each compound was 2500. The outputs were then ranked using both RosettaLigand energy function and ligand–protein interface scores to determine the binding position of lowest energy. The most energetically favourable docking positions were visualised using the PyMOL Molecular Graphics System Version 188.8.131.52 (Schrödinger).
Using primers designed against known sequences from chicken, cDNA fragments spanning the LBD of PR, AR, ESR1 and ESR2 were isolated and sequenced in the five species of parrots (kākā, kākāpō, kākāriki, kea and cockatiel) as well as the chicken and Japanese quail. For all receptors investigated, the amino acid sequences were identical in each different tissue type. All new sequence information as a result of the present study was sent to the National Centre for Biotechnology Information (NCBI) and has been attributed accession numbers (Table 2).
Molecular cloning of PR LBD
A cDNA fragment of 148 bp was isolated from kākāpō and the predicted translated sequence of this region of the kākāpō PR LBD was 49 amino acids in length (Fig. 1). A cDNA fragment was also amplified in the three other New Zealand parrots. There were three positions where the nucleotide differed between kākāpō and kākāriki and one nucleotide differed between kākā, kākāpō and kea (see Fig. S1, available as Supplementary Material to this paper). The predicted amino acid sequence was 100% identical across the five parrot species as well as with chicken. No tissue-specific differences in the sequences were observed.
A further sequencing attempt of kākāpō PR was undertaken upstream of the initial amplification region and a cDNA fragment of 650 bp was isolated (Fig. 2). The predicted translated kākāpō sequence was 216 amino acids in length. A fragment of same length was isolated from the other avian species studied. Sequence information beyond the 650-bp sequence at the N-terminal end was omitted due to poor quality. This section exhibited much more variation with differences at 36 nucleotide positions (Fig. S2). Alignment of this section of the PR LBD in the bird species showed 99% conservation across the four NZ parrot species and cockatiel, and 97% conservation compared with chicken (Fig. S3).
Molecular cloning of AR LBD
The cDNA fragment for the LBD of Kākāpō AR was 418 bp and the predicted translated sequence was 139 amino acids in length (Fig. 3).
Alignment of the LBD of kākāpō AR with the other four New Zealand birds exhibited 97% identity (Fig. S4). Comparison of the predicted amino acids showed 100% sequence identity when kākā, kākāpō and kea were compared and 99% identity with kākāriki (Fig. S5).
Molecular cloning of ESR1 LBD
When the LBD of ESR1 for kākāpō was amplified, a cDNA fragment of 615 bp was obtained and the predicted translated protein was 204 amino acids in length. Further sequencing of ESR1 LBD was undertaken generating a cDNA fragment of 266 bp and a predicted protein sequence consisting of 82 amino acids (Fig. 4). Alignment of the nucleotide sequences for the NZ parrot species and cockatiel showed nucleotide differences at 39 positions (Fig. S6). The greatest level of variation was seen when kākāpō was compared with kākāriki. Alignment of the 293 amino acids (Fig. S7) there were 10 positions where the amino acids were different.
Nucleotide sequence alignment of the NZ parrots with chicken and Japanese quail showed two indels that were three and four nucleotides in length (Fig. 5a). These sequence differences resulted in an extra eight amino acids at the C-terminal end of the receptor for the NZ parrots (Fig. 5b).
Molecular cloning of ESR2 LBD
A cDNA fragment of 251 bp was isolated from the kākāpō with a predicted translated amino acid sequence of 83 amino acids in length (Fig. 6). There were two tissue-specific nucleotide differences between the ovary and kidney in kākāpō (data not shown), but there were no differences in the predicted amino acid sequence. Alignment of the nucleotide sequences for the NZ parrot species and cockatiel showed nucleotide differences at nine positions (Fig. S8). The protein sequence was 100% identical across the five parrot species and this level of conservation decreased to 96% when compared with chicken (data not shown).
Modelling of 17β-oestradiol and phytoestrogens in kākāpō ESR1 LBD
The three-dimensional structure for kākāpō ESR1 was produced based on the human crystal structure (PDB entry 1A52). Within the LBD, there were several amino acids that differed to the human ESR1 LBD (Fig. S9). When the predicted docking position of 17β-oestradiol in the LBD of kākāpō ESR1 was compared with that observed in human, the orientation was found to be different, and thus the interactions between the side chains of amino acids and 17β-oestradiol were different (Fig. 7).
Simulations were also performed for several known phytoestrogens to see their binding position in the LBD of kākāpō ESR1. The phytoestrogen coumestrol was found to have an optimal binding position at a similar position to that for 17β-oestradiol (Fig. 8a). In contrast, daidzein had a binding position at a different orientation to 17β-oestradiol (Fig. 8b).
Phytoestrogens have also been isolated from NZ native plants. When podocarpinol and totarol were docked in the LBD (Fig. 8c, d), their orientation was more similar to that of daidzein than 17β-oestradiol.
The present study has identified unique sequence differences in the LBD of ESR1 for the parrot species investigated herein compared with the chicken. This information supports the hypothesis that exposure to phytoestrogens may have different dose-related effects in parrots compared with other avian species. Moreover, it is possible that these effects may provide a crucial link between diet and reproductive success. The present study represents the first reported sequences for steroid receptors in NZ parrots. In the present study, partial sequences spanning the LBD for four different steroid receptors, namely PR, AR, ESR1 and ESR2, from the NZ parrots kākā, kākāpō, kākāriki and kea, as well as the Australian cockatiel were isolated successfully. For each receptor, the sequence alignments showed a high level of conservation within the parrot species and the sequences were identical in all tissues with each species tested. Moreover, there was a high level of conservation compared with other avian species (e.g. chicken). However, an important exception to this was that indels were present in the ESR1 of parrots compared with the chicken.
The LBD of the steroid receptors investigated in the present study all exhibited a high level of conservation among the four NZ parrot species. In terms of the predicted amino acid sequences, PR was 100% identical in the first section sequenced and 99% conserved in the remaining sequence (Fig. S3). ESR2 was 100% identical for the five parrot species, whereas AR had only one amino acid difference in kākāriki. When these sequences were compared with other avian species using the NCBI Blastp tool, there was also a high degree of conservation. This is perhaps unsurprising due to the high level of conservation known to be present in nuclear receptor LBD regions.
However, the greatest level of sequence variation within the steroid receptors studied was for ESR1. The 39 nucleotide differences translated into 10 positions where the amino acids differed. When the ESR1 sequence for the NZ parrots was compared with that for chicken (NM_205183) and Japanese quail (AF44296.1), the alignment showed a three- and four-nucleotide indel (see Fig. 5a). This resulted in the sequences for kākāpō, kākā, kākāriki, kea and cockatiel being eight amino acids longer. These differences were located at the C-terminal end of the protein (Fig. 5b). When Blastp was used to identify similar protein sequences in other avian species, only one, the predicted sequence for budgerigar (Melopsittacus undulates; XP_005150137; C. E. J. David, unpubl. obs.), was found to contain the extra eight amino acids at the C-terminal end. This suggests these additional amino acids may be specific to parrots. This difference is likely to lead to an alteration in the shape of the binding pocket for the ligand and, in turn, may well affect the binding affinity between the receptor and steroidal compounds between the different avian species.
As stated earlier, oestrogens are known to be critical regulators for reproductive processes as well as being important for influencing many non-reproductive processes. Therefore, any change to the oestrogen-binding region of the receptor could affect these processes (in either a positive or detrimental manner) by influencing their reproductive behaviour. For example, because ESR1 is intrinsically linked with vitellogenesis, the species differences found for this receptor sequence could potentially affect this process (Li et al. 2014).
Hence, by using the known three-dimensional structure of ESR1 for human, the LBD of kākāpō ESR1 was simulated. The differences in amino acids were shown to alter the optimal binding position for 17β-oestradiol (Fig. 7). This supports the notion that oestrogens could interact with the receptor differently in parrots, including kākāpō, compared with other species. The different binding positions and orientations of known phytoestrogens (Fig. 8) also support this. It should be noted that species-specific differences that occur outside the LBD of ESR1 may also affect the binding affinity of 17β-oestradiol to its receptor. Obtaining crystal structures of nuclear receptors in their native fold state has proved technically challenging (Rastinejad et al. 2013); however, new high-resolution structures of full-length nuclear receptors are beginning to emerge and provide snapshots of their complex physiologically functional states. Analyses reveal that the mere act of the ligand binding into the hydrophobic pocket of its nuclear receptor alters the quaternary structure of the receptor, which may expose non-conserved areas within the DBD–LBD interfaces and alter coregulatory protein interactions that are unique to each receptor (Khorasanizadeh and Rastinejad 2016). Thus, sequence differences at sites distant from the LBD may affect binding affinities as well as downstream signalling events, but testing this would require sophisticated modelling methods and is outside the scope of the present study.
Fidler et al. (2008) hypothesised that there is a link between the ingestion of phytoestrogens and the expression levels of the egg yolk protein gene. The expression of oestrogen receptor isoforms in the liver of the avian species is not inconsistent with the aforementioned hypothesis. Therefore, to gain a better understanding of the reproductive process of kākāpō and related NZ as well as other parrot species, the complete sequence of ESR1 needs to be obtained. From this, a representation of the three-dimensional structure and potential binding capabilities of this receptor could be considered.
With regard to putative links between plant hormonal activity and reproductive success in parrots, very little information is currently available. Moreover, given the major differences in breeding behaviours between parrots, a useful starting point in investigating such links may be to use a cell-based bioassay transfected with the parrot ESR1 gene and examine extracts of plants that are known to be ingested by parrots. In the case of kākāpō, several native plants have been identified to be part of their diet (Atkinson and Merton 2006; Wilson et al. 2006; Horrocks et al. 2008) or are abundant in areas where they graze (Butler 2006). Importantly, these plants have oestrogenic activity, including Rimu (Dacrycarpus cupressinum), kiokio (Blechnum novae-zelandiae), southern beech (Nothofagus solandri) and miro (Prumnopitys ferruginea). When extracts from these plant species were assessed for oestrogenicity in a bioassay using yeast transfected with human ESR1, all were found to have activity, with the highest levels detected in kiokio (Davis 2013). These studies by Davis (2013) also confirmed the presence of oestrogenic activity in podocarpic acid and podocarpinol, two key compounds present in rimu (Fidler et al. 2000), as well as totarol present in the native totara (Podocarpus totara) (Terasaki et al. 2007). However, the chemical structures of the oestrogenic materials of most NZ native plants are not known. Therefore, the identification of plant chemicals interacting with a parrot-based ESR1 detection system together with information regarding the plant grazing behaviours of parrots may provide new insights into the conservation of those considered to be in decline. In turn, this may have potential future application for exploring diet supplementation with relevant plant extracts to increase reproductive success in endangered NZ parrots.
Because the kākāpō is one of the most endangered species in the world, any information related to its reproductive strategy is extremely important. The present study compared the LBD of steroid hormones between parrots and other avian species. The greatest variation in sequence was shown for ESR1. This finding adds support to the notion that the ESR1 LBD of parrots may respond differently to oestrogenic activities in native NZ flora during times of intermittent masting. In turn, this may provide important information for generating bioassay systems to test the steroidogenic activity of NZ plant species.
The authors acknowledge funding assistance from the New Zealand Royal Society Pickering Award (to Kenneth P. McNatty). The authors also thank the New Zealand Department of Conversation and Dr Andrew Fidler for supplying tissues, and Dr Murray Williams for his expert advice and assistance with tissue samples.
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