Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

Accepted Manuscript Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

Ruidong Zhang, Yanan Yin, Long Sun, Peng Yan, Yongkang Zhou, Rong Wu, Xiaobing Wu PII: DOI: Reference:

S0378-1119(17)30279-2 doi: 10.1016/j.gene.2017.04.028 GENE 41877

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Gene

Received date: Revised date: Accepted date:

6 December 2016 4 January 2017 12 April 2017

Please cite this article as: Ruidong Zhang, Yanan Yin, Long Sun, Peng Yan, Yongkang Zhou, Rong Wu, Xiaobing Wu , Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi: 10.1016/ j.gene.2017.04.028

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ACCEPTED MANUSCRIPT

Title Page Title: Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

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Authors:

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Ruidong Zhanga, Yanan Yina, Long Suna, Peng Yana, Yongkang Zhoub, Rong Wub, Xiaobing Wua*

a

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Affiliations:

Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province,

Alligator Research Center of Anhui Province, Xuanzhou 242000, China

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b

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College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000;

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*Correspondence author:

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Dr. Xiaobing Wu

College of Life Sciences, Anhui Normal University, 1 Beijing East Road, Wuhu, Anhui Province

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241000, People’s Republic of China.

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Tel: +86 13855304381; Fax: +86 5533836837 Email: [email protected] Other authors:

Ruidong Zhang, Email: [email protected]; Yanan Yin, Email: [email protected]; Long Sun, Email: [email protected]; Peng Yan, Email: [email protected]; Yongkang Zhou, Email: [email protected]; Rong Wu, Email: [email protected].

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ACCEPTED MANUSCRIPT Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

Abstract

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Estrogens play critical roles in reproductive physiology via estrogen receptors (ESRs) in

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vertebrates, including reptiles. Chinese alligator (Alligator sinensis) is an endemic and endangered

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reptile species in China. In the present study, we cloned ESR2 gene from the ovary using rapid amplification of cDNA ends (RACE), investigated the spatial expression of ESRs in various

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tissues and temporal expression of ESRs in the pituitary glands during the reproductive cycle in

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Chinese alligators by quantitative real-time PCR (qPCR). Bioinformatics and phylogenetic analysis of deduced ESR2 protein were also performed. The full-length cDNA of the ESR2 is 1647

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bp in length, with an open-reading frame encoding 548 amino acids. The bioinformatics analysis

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indicated that the deduced amino acid sequence of alligator ESR2 was highly conserved with that of other vertebrate species. In addition, compared to human ESR2, the 14 amino acids in the

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alligator ESR2 that are essential for specific recognition of estradiol are entirely conserved. The

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phylogenetic analysis showed that alligators were more closely related to birds than to other reptiles. The results of qPCR showed that the tissue distribution patterns of both ESR subtype mRNAs appeared to be different. In male tissues, the highest mRNA level of both ESRs is in the liver. While in female tissues, ESR1 and ESR2 showed the highest mRNA level in the hypothalamus and pituitary gland, respectively. During the female reproductive cycle, the expression level of ESR1 mRNA increased from the initial post-hibernation period to the reproductive period, reached its peak in the reproductive period, and then decreased in the autumn

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ACCEPTED MANUSCRIPT active period and hibernation period. Conversely, the highest transcription level of ESR2 was observed in the hibernation period.

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Keywords: ESR2, Alligator sinensis, cloning, 3D model, gene expression, reproductive cycle.

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1. Introduction

The vital functions of estrogen on the growth and development of vertebrate had been

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inconsistently proven by previous studies, such as the neurogenesis, learning and memory, energy

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homeostasis, auditory processing, social communication and so on –( Kwan et al., 1996; McEwen and Alves, 1999; Takaoka et al., 2002; Barha and Galea, 2010; Verderame and Limatola, 2010;

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Cornil et al., 2012; Remage-Healey et al., 2012; Bailey et al., 2013; McCarthy and Nugent, 2013;

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Bless et al., 2014; Arevalo et al., 2015; Iran-Nejad et al., 2015; Zahedi et al., 2015; Shiga et al., 2016). In common, the adult reproductive function was considered to be controlled through a

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negative feedback loop by gonadotropins, androgens and estrogens that involved the

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hypothalamus, pituitary gland and gonad known as the hypothalamus–pituitary–gonadal axis (HPG) (Tilbrook and Clarke, 2001; Boon et al., 2010; Kwakowsky et al., 2012). Estrogen actions are mediated by two forms of estrogen nuclear receptor that belong to the steroid nuclear receptor superfamily. Two distinct subtypes of estrogen receptors (ESRs) have been reported ( Green et al., 1986; Kuiper et al., 1996), including estrogen receptor alpha (ERα/ESR1) and estrogen receptor beta (ERβ/ESR2). ESRs were regarded as ligand-dependent transcriptional factors as well (Tetel

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ACCEPTED MANUSCRIPT et al., 2009). In 1986, ESR1 was cloned and sequenced from human for the first time ( Green et al., 1986). And then, ESR2 was cloned and sequenced until 1996 from rat ( Kuiper et al., 1996). ESR1 and ESR2 are transcribed from two different genes but share a certain degree of homology and bind to estradiol with a similar affinity (Mueller et al., 2004). However, the two

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receptors exhibit clear differences in their ligand-binding domains, transactivation of target genes

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and tissue-specific expression pattern and histological distribution (Paech et al., 1997; Cao and

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Patisaul, 2013). ESR1 plays an indispensable role in maintaining normal reproduction that is undisputed. ESR1-knockout female rats are completely infertile (Rumi et al., 2014). By contrast,

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the role of ESR2 in the maintenance of normal reproductive function has not been fully elucidated

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(Koehler et al., 2005; Sugiyama et al., 2010). ESR2-knockout female mice are subfertile compared with wild type (Couse et al., 2005). Due to the delay of follicular maturation and rupture, the

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ESR2-knockout female mice show fewer ovulation and pregnancies (Dupont et al., 2000; Inzunza

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et al., 2007).

Chinese alligator (A. sinensis) is a rare and endemic species in China. It is listed as Critically

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Endangered by the IUCN Red List. There are less than 150 Chinese alligators in the wild

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nowadays. It is of great significance for the recovery of wild alligator population to actively carry out the researches of reproductive biology. Chinese alligator is a typical seasonal breeder and its reproductive cycle is affected by hormonal and local factors, including estrogens (Ogawa et al., 1998; Hamlin et al., 2014). However, researchers pay less attention on the study of mechanism of the regulation of reproductive hormones. Its reproductive cycle can be divided into different periods, including autumn active period (August - October), hibernation period (from November to next year February), initial post-hibernation period (March), spring active period (April - May)

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ACCEPTED MANUSCRIPT and reproductive period (June - July) (Chen et al., 2003; Zhang et al., 2015b). Chinese alligators suffer from the low temperature and lack of food in hibernation period and have aggressive and mating behaviors in reproductive period. Eating lots of food during autumn and spring active periods are necessary to prepare for the coming hibernation and reproductive periods (Chen et al.,

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2003). In previous study, we had cloned and sequenced the full-length cDNA of ESR1 from

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Chinese alligator, as well as reported the ESR1 mRNA expression levels changes during its

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hibernation and reproductive periods (Zhang et al., 2016). However, there is no other study about ESRs in this species.Nowadays, full-length cDNA of ESR2 has been cloned in many vertebrate

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species including human (Strausberg et al., 2002), common starling (Bernard et al., 1999),

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American alligator (Katsu et al., 2010), tropical clawed frog (Takase and Iguchi, 2007) and zebrafish (Menuet et al., 2002). However, there are fewer researches on full-length cDNAs of

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ESR2 in reptiles. Specific to the full-length cDNA sequences of ESR2 in crocodilian species, only

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American alligator (Alligator mississippiensis) was reported so far (GenBank No. NM001287264). To further understand the molecular endocrinology of Chinese alligator, we isolated and

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sequenced ESR2 cDNA in the present study. In addition, the relative expression of ESR cDNAs

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quantitatively in different tissues (spatial variation) and during the reproductive cycle (temporal changes) was described by using the quantitative real-time PCR (qPCR).

2. Materials and methods 2.1. Sample preparation Twelve mature alligators (10 females and 2 males) over 15 years old, were collected from the Anhui Research Center for Chinese Alligator Reproduction Center (ARCCAR) during the

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ACCEPTED MANUSCRIPT different phases of the reproductive cycle: March for initial post-hibernation period, the end of May for spring active period, July for reproductive period, the end of August for autumn active period, and January for hibernation period (Supplementary table 1). Meanwhile, all experimental activities strictly complied with the rigorous permitting process by the State Forest Administration

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(SFA) to reduce injuries on endangered species as much as possible. Two female alligators for

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each period and two male alligators only in January were collected and killed with a lethal dose of

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Nembutal, and various tissues were isolated and immediately preserved in RNAstore (Tiangen Biotech, Beijing, China) and then stored at -80 ℃. Different tissues of Chinese alligator were used

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in this study, including the hypothalamus, pituitary gland, gonad, liver, lung, kidney and pancreas.

2.2. RNA extraction and first-strand cDNA synthesis

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Total RNA was extracted from tissues by using a RNAprep Pure Tissue Kit (Tiangen Biotech,

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Beijing, China) under a standard procedure. RNA purity and integrity were determined by TGem spectrophotometer (Tiangen Biotech, Beijing, China) analyses at 260/280 nm, then screened by

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2% agarose gel electrophoresis. RNA samples were dissolved in RNase-free water at the

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concentration of 1 μg/μl and reverse transcribed by using a PrimeScriptTM RT reagent Kit (TaKaRa, Dalian, China) in a 20 μl reaction volume containing 4.0 µl of 5 × PrimeScript Buffer 2, 2.0 µl of 5 × gDNA Eraser Buffer, 1.0 μl of gDNA Eraser, 1.0 μl of RT Primer Mix, 1.0 μl of PrimeScript RT Enzyme Mix I, 1.0 μl of total RNA, and the final volume was adjusted using RNase-free water. Thermal cycling was carried out for 15 min at 37 ℃, then 5 s at 85 ℃. The RT products were stored at -20 ℃ until used.

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ACCEPTED MANUSCRIPT 2.3. Molecular cloning of A. sinensis ESR2 The isolation of complete ESR2 sequence from A. sinensis was performed with PCRs and 5’/3’ RACE. According to the high conserved regions of the ESR2 cDNA sequence from A. mississippiensis (NM001287264), we designed one primer pair, ESR2F and ESR2R (Table 2).

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With the very primers, a 780-bp fragment of ESR2 cDNA was amplified by using the template of

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the first strand cDNA, which was isolated and synthesized from the total RNA of ovary. PCR was

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performed in a 30 μl reaction mix at the following conditions: 94 ℃, 5 min, 1 cycle; 94 ℃, 30 s, 56 ℃, 60 s, 72 ℃, 90 s, 35 cycles; and 72 ℃, 10 min. PCR products were analyzed by 1.0 %

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agarose gel electrophoresis and purified utilizing a TIANgel Midi Purification Kit (Tiangen

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Biotech, Beijing, China). The target fragment was cloned into pMD19-T vector and sequenced ultimately.

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The 5’- and 3’-ends of the ESR2 cDNAs were amplified using rapid amplifcation of cDNA

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ends with the 5’/3’ rapid amplification of cDNA ends (RACE) kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. All sequences of the cloned cDNA were confirmed

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by the BLAST Program of National Center for Biotechnology Information (NCBI).

2.4. Bioinformatic and Phylogenetic analysis The prediction of open reading frame (ORF) on ESR2 was conducted by using the BLAST Program of NCBI (http://www.ncbi.nlm.nih.gov/blast). The amino acid sequence, protein molecular weight (MW) and isoelectric point (PI) of ESR2 were predicted using ExPASy (http://web.expasy.org/protparam/). The homology between ESR2 amino acid sequence of Chinese alligator and that of other vertebrate species were conducted with DNAMAN7 program.

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ACCEPTED MANUSCRIPT The deduced full-length amino acid sequences of ESR2 were aligned using the ClustalX2 program. Any gaps were removed from the sequences using the BioEdit program. Other ESR2 sequences used in this study were collected from NCBI (Table 1). A phylogenetic tree was constructed with the neighbor-joining (NJ) method by the MEGA6 program. The ESR1 sequence

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of Chinese alligator was used as an outgroup (GenBank Accession No. KU356777). The reliability

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of the tree was assessed with 1000 bootstrap replications.

2.5. Quantitative real-time PCR

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Relative ESR1 and ESR2 mRNA expression levels of A. sinensis were measured by qPCR in

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the Bio-Rad iQTM5 Multicolor Real-Time PCR Detection System (Bio-rad, Hercules, CA) with SYBR Premix Ex Taq II (Tli RnaseH Plus) Kit (TaKaRa, Dalian, China). Amplification reactions

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were carried out in triplicate and each 20 μl reaction volume contained 1.6 µl of cDNA solution,

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0.4 µl of forward and reverse primers, 10 µl of SYBR Premix Ex Taq II and 7.6 µl of double-distilled water. The qPCR conditions were as follows: 5 min at 94 ℃, 40 cycles of

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denaturation at 94 ℃ for 30 s and annealing and extension at 58 ℃ for 30 s. The ribosomal protein

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L8 (RPL8) was selected as an internal reference gene and the expression level of RPL8 was used to normalize the qPCR results for each gene. All primers used in this study are shown in Table 2 (the primer pairs of ESR1L/ESR1H and RPL8F/RPL8R had been used in the previous studies (Katsu et al., 2004; Zhang et al., 2016)). Negative controls (without a cDNA template) were included in each qPCR experiment. Melting curves were analyzed to verify that only a single PCR product was amplified for each pair of primers. Product purity was confirmed by 3.0 % agarose gel electrophoresis. Standard curves were used to calculate amplification efficiencies, which were

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ACCEPTED MANUSCRIPT constructed by serial dilutions (1:10) of cDNA. The cycle threshold (Ct) value was automatically determined by the Bio-Rad iQ5 Real-time PCR Detection software. The relative levels of expression for ESR1 and ESR2 were calculated relative to RPL8 using the 2-ΔΔCt method. Data were analyzed using Student’s t-test and one-way

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ANOVA on SPSS 19.0. The significance level was set at P < 0.05.

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2.6. Construction of 3D Model

The homology modeling for the ESR2-LBD (the ligand-binding domain, LBD) and the

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interaction potentials between ligand and the LBD receptor model were performed using

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Swiss-Model's online automated protein structure homology modeling server (Peitsch et al., 1995; Arnold et al., 2006; Kiefer et al., 2009). The homology model of ESR2-LBD was constructed on

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the basis of the crystal structure of human ESR2-LBD with estradiol (PDB ID: 2j7x.A) in Protein

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Data Bank (PDB). The predicted model was visualized and overlayed with the human ESR2 for

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3. Results

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comparison using the program of Pymol (DeLano, 2002).

3.1. Cloning and sequence analysis of Chinese alligator ESR2 By using the standard PCR, a DNA fragment was amplified from alligator ovary RNA, which showed significant sequence similarities to the ESR2 of A. mississippiensis. The full-length ESR2 cDNA sequence from Chinese alligator was obtained finally and submitted to GenBank (GenBank Accession No. KX267840). The ESR2 cDNA is composed of 1647 nucleotides and encodes a predicted 548 amino acids with a calculated MV of 61.4 kD and a PI of 8.44 (Figure 1). The

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ACCEPTED MANUSCRIPT sequence, as defined by Krust et al. (1986), can be divided into A/B, C, D and E/F domains based on its sequence homology with other steroid hormone receptors. The ESR1 and ESR2 of Chinese alligator shared 16.3% identity in the A/B domain, 95.5% identity in the C domain, 37% identity in the D domain, and 51.2% identity in the E/F domain (Figure 2A). Thus, the C domain (the

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DNA-binding domain, DBD) is highly conserved and the E/F domain (LBD) is also conservative

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between the ESR1 and ESR2. Comparing with multiple vertebrate ESR2, we found that both

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DBDs and LBDs are highly conserved (100 - 87.9%, 53.0 - 99.5%) (Figure 2B). The genetic similarity among vertebrates varied from 0.488 to 0.990, of which the Chinese alligator homology

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with the American alligator was the highest with a value of 0.990. The NJ tree for full-length

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amino acid sequences of the ESR2 indicated that crocodilians were more closely related to birds

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than to other reptiles (Figure 4).

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3.2. Software-based construction of 3D model of the ligand-binding domain of Alligator sinensis ESR2

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We analyzed the predicted protein structure of ESR2 protein of Chinese alligator using

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SWISS-MODEL, and made a comparison with human ESR2 (Figure 5). The LBD is highly conserved (92.3% identity) between alligator and human (Figures 3 and 5A). The 14 amino acids that are essential for specific recognition of estradiol are entirely conserved (Figures 3 and 5B), suggesting the alligator ESR2 plays the similarity manner as the human ESR2.

3.3. Transcription/mRNA levels analysis of estrogen receptors in multiple tissues The mRNA levels of both estrogen receptors in multiple tissues, which were from female and

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ACCEPTED MANUSCRIPT male adult alligators in January, were determined by qPCR. ESR1 and ESR2 mRNAs are widely distributed in female and male tissues as shown in Figure 6. In male tissues, the levels of ESR2 mRNA are significantly higher than that of ESR1 mRNA in the hypothalamus, pituitary gland and liver (P < 0.01). In female tissues, the levels of ESR2 mRNA also appear higher than that of ESR1

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mRNA in the pituitary gland and ovary (P < 0.01), while just the opposite in the hypothalamus,

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liver, lung and kidney. Significant differences in expression levels of ESR are also observed in the

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tissues between females and males, eg hypothalamus, pituitary gland, gonad and liver (P < 0.05)

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(Supplementary figure 1).

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3.4. Changes in the gene expression of estrogen receptors in pituitary gland during different stages of reproductive cycle

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We also analyzed the mRNA levels of ESRs in the pituitary gland during the female

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reproductive cycle. As described by Chen et al. (2003) andZhang et al. (2015b), we collected five stages of reproductive cycle: initial post-hibernation period, spring active period, reproductive

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period, autumn active period and hibernation period. Analyzing the mRNA levels of ESR1, we

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observed the mRNA expression level increased from the initial post-hibernation period to the reproductive period, reached its peak in the reproductive period, and then decreased in the autumn active period and hibernation period. The mRNA expression of ESR1 in the reproductive period was significantly higher than that in other periods (P < 0.05). For ESR2 mRNA expression, we found the mRNA expression level increased from the initial post-hibernation period to the spring active period, then decreased in the reproductive period, reached its bottom in the reproductive period, and then increased in the autumn active period and hibernation period. The highest mRNA

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ACCEPTED MANUSCRIPT level of ESR2 is in the hibernation period. The mRNA expression of ESR2 was significantly higher in the hibernation period compared with other periods (P < 0.05) (Figure 7).

4. Discussion

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Chinese alligator is a seasonal breeder, with a reproductive period during summer and a

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hibernation period during winter (Chen et al., 2003). In 2010, a full-length cDNA sequence for the

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ESR2 was first reported from crocodilian, American alligator, A. mississippiensis (Katsu et al., 2010). Since then, no additional sequence has been reported in crocodilian yet. In the present study,

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we report the full-length cDNA sequence of ESR2 in A. sinensis for the first time and deduced the

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amino acid sequence based on the gene sequence. The mRNA expression of ESR1 and ESR2 in different organs from male and female alligators, was investigated by qPCR. In addition, their

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reproductive cycle as well.

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gene expression patterns in the pituitary glands were determined by qPCR during the female

The full-length cDNA cloning for Chinese alligator ESR2 shows a high level of similarity

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with the ESR2 identified in several reptiles and birds, such as American alligator (Katsu et al.,

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2010), soft-shell turtle and common starling (Bernard et al., 1999). In Chinese alligator, the identities of deduced amino acid sequences between ESR1 and ESR2 were 43.8% for the complete sequence, 95.5% for the DBD and 51.2% for the LBD (Figure 2). According to the Figures 2 and 3, interestingly, the highest identities domain of ESR2 is the DBDs among these vertebrates. These DBDs also contained several conserved motifs and elements (the zinc-finger motifs, P-box, D-box and PKA), which are essential to precise reorganization (Figure 3) (Vanacker et al., 1999). These structural similarities suggest that the alligator ESR2 binds similar

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ACCEPTED MANUSCRIPT estrogen responsive elements compared to other vertebrate ESR2. Sequence identities among LBDs of Chinese alligator and other vertebrate animals are also relatively high (56.6–99.5%). On the amino acids of crocodilian LBDs, Ala of American alligator was replaced by Ser in Chinese alligator. Furthermore, the 14 amino acid residues and AF-2 (Figures 3 and 5B), which are

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associated with estradiol binding, are almost completely conserved in the LBD from fish to

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mammals (Ekena et al., 1996; Brzozowski et al., 1997; Baker et al., 2009). The high degree of

responsive elements much like other vertebrate ESR2s.

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conservation for these key residues suggests the alligator ESR2 binds estradiol and estrogen

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The phylogenetic analysis revealed that crocodilians were the sister group to birds. This

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result was consistent with the traditional grouping of birds and crocodilians into the Archosauria (Katsu et al., 2010). Phylogenetic analysis of vertebrates by the complete mitochondrial genome

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sequences, follicle-stimulating hormone β subunit and B-cell activating factor amino acid

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sequences, had also supported this traditional grouping (Wu et al., 2003; Zhang et al., 2015a; Zhang et al., 2015b). However, the result of phylogenetic analysis of ESR1s showed that

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2016).

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crocodilians were the sister group to the turtles and closely related to the birds (Zhang et al.,

The qPCR analysis revealed that the mRNA of ESRs was expressed in both male and female alligators, and it showed obvious tissue-specific gene expression patterns, implying multi-functionality of ESRs in different tissues. In this study, greater expression both in ESR1 and ESR2 could be observed in some female tissues, which are induced to associate with reproduction, such as hypothalamus, pituitary gland, ovary and liver. Moderate levels of both ESR genes were found in hypothalamus, pituitary gland and testis tissues from males, but high levels of gene

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ACCEPTED MANUSCRIPT expression was showed in the liver, which was consistent with the report of Esterhuyse et al. (2010). Higher expression levels of both ESR genes in the tissues (hypothalamus, pituitary gland, gonad and liver) of male and female alligators that agreed with the results previously found in the killifish, cod, hagfish, four-striped rat snake, pigeon, European starling and rat (Kuiper et al., 1997;

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Bernard et al., 1999; Katsu et al., 2010; Nagasawa et al., 2014; Zhang et al., 2014; Cotter et al.,

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2015; Nishimiya et al., 2016). The ESRs mRNA levels in the lung, kidney and pancreas were low.

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These results further indicated that ESR genes possessed many functions, but the main function was in the regulation of reproduction. However, we found that the expression levels of ESRs

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mRNA had significant differences in the reproductive related tissues (hypothalamus, pituitary

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gland, gonad and liver) between females and males. This might suggest that the ESRs mediated different reproductive functions of estrogenic actions in males and females. In addition, we found

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that the mRNA level of ESR2 was higher than ESR1 in the pituitary gland of male and female

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alligators during hibernation period. This result indicated that ESR1 might mainly mediate estrogenic reproductive function in the pituitary gland, which was supported by our previous study

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about Chinese alligator (Zhang et al., 2016). By contrast, ESR2 might mediate estrogenic several

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non-reproductive functions in hibernation period, such as modulating anxiety-like behavior and activity of the hypothalamic–pituitary–adrenal axis (HPA) (Krezel et al., 2001; Lund et al., 2006; Weiser et al., 2009). In male mice, ESR2 could inhibit aggressive behavior that was in contrast to the role of ESR1 (Ogawa et al., 2000; Nomura et al., 2002). In male goldfish and mosquitofish, ESR2 mRNA expression levels in liver lacked significant changes in response to estradiol. In contrast, ESR1 might be the primary mediator of estradiol actions on the liver associated with the induction of vitellogenins mRNA expression during the reproductive period (Pomatto et al., 2011;

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ACCEPTED MANUSCRIPT Huang et al., 2013). These data might be a reason for the expression levels of male alligator ESR2 also significantly higher than ESR1 in the hypothalamus and liver during the hibernation period, which were similar to the results of Socorro et al. (2000) and Verderame and Limatola (2010). However, in female alligators, the mRNA expression level of ESR1 was higher than that of ESR2

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in the hypothalamus and liver, which was consistent with the previous reports in the cod

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(Nagasawa et al., 2014), Italian wall lizard (Verderame and Limatola, 2010), leopard gecko (Endo

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et al., 2008) and bovine (Panin et al., 2015).

Meanwhile, the expression levels of the two ESRs mRNA in the pituitary gland were

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analyzed by the qPCR method in the study. It was the first one report on the female Chinese

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alligators during the reproductive cycle. The result showed significant changes were observed on the two ESRs mRNA expression levels, which suggested that ESRs might play some roles in the

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control of reproduction in Chinese alligators. The level of ESR1 mRNA was clearly raised during

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the reproductive period (July) and decreased in the autumn active period (end of August). Interesting, ESR2 showed the highest levels of mRNA expression in the hibernation period

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(January), but not the reproductive period. These results suggested that the regulating function of

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ESRs in the pituitary gland were very complex during the reproductive cycle. Both ESRs mediated the negative feedback of estradiol on secretion of luteinizing hormone (LH) at the level of the pituitary gland in mammals (Fink, 1988; Schwartz, 2000; Arreguin-Arevalo et al., 2007). Estrogens maintained LH secretion at low levels during estrus, metestrus, diestrus and proestrus morning in rats and mice through their negative feedback regulation (Fink, 1988). ESR1 and ESR2 might mediate partially the positive feedback of estradiol on LH secretion by increasing the expression of gonadotropin-releasing hormone (GnRH) receptors and only ESR1 was thought to

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ACCEPTED MANUSCRIPT be involved in the negative feedback of estradiol on secretion of follicle-stimulating hormone (FSH) in ewes (Arreguin-Arevalo et al., 2007). The roles of ESR1 and ESR2 within a particular neural network might be synergistic or antagonistic. For example, ESR1 enhanced anxiety-like and aggressive behaviors but ESR2 suppressed them (Handa et al., 2012). This could be one of the

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reasons for low ESR2 mRNA levels in reproductive period, when high levels of estrogen and

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aggressive behavior were present (Chen et al., 2003). In the rodent, ESR2 and ERβ2 (one of ESR2

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splice variants) levels showed an increase after ovariectomised (OVX) (Brinton and Wang, 2004; Lund et al., 2005). Similar result was also reported in female Wistar rats that OVX had resulted in

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ESR2 and ERβ2 mRNA increases in the pituitary gland, whereas ESR1 mRNA expression was not

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affected (Tena-Sempere et al., 2004). These data suggested that ESR2 and ERβ2 mRNA expression increased during the period of devoid of endogenous estrogen. In this study, the highest mRNA

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expression levels of ESR2 were observed in hibernation period, which might be resulted by the

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lack of gonadal hormone. And higher ESR2 mRNA levels during spring and autumn active periods also might be due to decreases in estrogen levels. However, changes of ESR1 expression levels

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during reproductive cycle were consistent with the previous studies in cod (Nagasawa et al., 2014)

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and Italian wall lizard (Verderame and Limatola, 2010), of which the highest expression of ESR1 were observed during the reproductive period. This result further illustrated that ESR1 in the pituitary gland mainly mediated estrogenic reproductive function.

5. Conclusions We cloned and sequenced ESR2 cDNA from Chinese alligator. This is the first report of the full-sequence information of ESR2 on this endangered species. The phylogenetic tree revealed

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ACCEPTED MANUSCRIPT there have a close relationship between the crocodilians and birds. The results of the sex- and tissue-specific expression and mRNA levels have some changes during the reprodutive cycle of both ESR subtypes, which provided a frame work for better understanding of physiological significance of these groups of receptors in reptiles. These data pave the way for future

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investigations regarding the basic endocrinology and molecular biology of non-mammalian steroid

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hormone receptors. Furthermore, this study provides an important basis for further studies on

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specific functions and mechanisms of actions of ESRs in juvenile and adult Chinese alligators, such as gonadal development, sex differentiation, both regulation and seasonal variation in

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hormonal.

Abbreviations

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ESRs: Estrogen receptors; ERα/ESR1: Estrogen receptor alpha; ERβ/ ESR2: Estrogen receptor

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beta; cDNA: DNA complementary to RNA; mRNA: Messenger RNA; PCR: Polymerase chain reaction; RT: Reverse transcription; qPCR: Quantitative real-time PCR; 3D: Three dimensional;

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HPG: Hypothalamus–pituitary–gonadal axis; IUCN: International Union for Conservation of

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Nature; RACE: Rapid amplification of cDNA ends; NCBI: National Center for Biotechnology Information; ORF: Open reading frame; MW: Molecular weight; PI: Isoelectric point; RPL8: Ribosomal protein L8; PDB: Protein Data Bank; DBD: DNA-binding domain; LBD: Ligand-binding domain; PKA: Protein kinase A; AF-2: Activation function-2; LH: Luteinizing hormone; GnRH: Gonadotropin-releasing hormone; FSH: Follicle-stimulating hormone; OVX: ovariectomy.

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ACCEPTED MANUSCRIPT Conflict of interest The authors declare that they have no competing interests.

Authors’ contributions

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Xiaobing Wu designed the study. Ruidong Zhang, Yanan Yin and Long Sun performed the

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experiments and data analysis. Yongkang Zhou and Rong Wu contributed materials. Ruidong

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Zhang wrote the manuscript. Peng Yan revised the manuscript. All authors read and approved the

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final manuscript.

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Acknowledgements

We would like to thank the staff of Anhui Research Center for Chinese Alligator Reproduction

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Center, who assisted in the collection of samples. This study was funded by the National Natural

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Science Foundation of China (NSFC, Grant No. 31272337, 31472019) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133424120001).

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The funders had no role in study design, data collection and analysis, decision to publish, or

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preparation of the manuscript.

Ethics approval

All experimental procedures adhered to the Animal Care and Use Committee of Anhui Normal University.

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Legends Figure 1 The nucleotide and deduced amino acid sequences of Chinese alligator ESR2. The numbers on the left refer to the position of the nucleotides and the amino acids.

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Figure 2 Domain structure of Chinese alligator ESRs and homology with ESR2 from other vertebrate species. Percent homology of the functional domains relative to the ESR2 is depicted. The functional A/B to E/F domains are schematically represented with the numbers of amino acid

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residues indicated. A, Comparison of Chinese alligator ESR1 with Chinese alligator ESR2. B,

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Domain structure of ESR2 in Chinese alligator, and identity with Alligator mississippiensis,

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Pelodiscus sinensis, Sturnus vulgaris, Homo sapiens, Xenopus laevis and Danio rerio ESR2s.

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Figure 3 Aligned deduced amino acid sequences of Chinese alligator ESR2 with that of other

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Figure 4 Neighbor-joining phylogenetic tree of vertebrate ESR2 amino acid sequences.

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Values are indicated at branch nodes. The scale bar represents 0.1 substitutions per site.

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Figure 5 ESR2 structure analysis. A, overlap of 3D model of Chinese alligator ESR2 (green) with human ESR2 (orange). The 3D model of Chinese alligator ESR2 with estradiol (red) was superimposed on human ESR2. There is excellent overlap. (Crystal structure accessions, PDB: 2j7x). B, Predicted binding modes obtained from the docking simulation analysis of estradiol for the ESR2-LBD. The 14 amino acid residues are contact with estradiol.

Figure 6 Tissue specific expression of ESR1 and ESR2 in adult Chinese alligators. Values are

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significance of differences in the levels of expression of ESR1/ESR2 mRNA was determined by

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ANOVA followed by Student’s t-test. Comparing the expression between ESR1 and ESR2, the “*”

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Figure 7 Relative expression of ESR1/ESR2 mRNA in the pituitary gland of Chinese alligator during different periods of the reproductive cycle. The expression levels of

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calculated by the 2-ΔΔCt method, are presented in arbitrary units. Values are the means ± SEM. The significance of differences in the levels of expression of ESR1/ESR2 mRNA was determined by

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ANOVA followed by Student’s t-test. Mean values bearing different letter superscripts are

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GenBank ID

Alligator sinensis Alligator mississippiensis Pelodiscus sinensis Chelonia mydas Pseudemys nelsoni Anolis carolinensis Bradypodion pumilum Plestiodon finitimus Elaphe quadrivirgata Protobothrops flavoviridis

KX267840 NP_001274193 XP_006134140 XP_007068065 BAJ15430 XP_008124047 BAU36957 BAU36959 BAJ15428 BAJ15427

Geospiza fortis Zonotrichia albicollis Sturnus vulgaris

XP_005417994 XP_014125903 AAD56593

Columba livia Homo sapiens Mus musculus Bos grunniens Sus scrofa Xenopus laevis Xenopus tropicalis Danio rerio Oryzias latipes Lepomis macrochirus Oncorhynchus mykiss

NP_001269770 AAV31779 AAI45330 AHM88215 NP_001001533 NP_001124426 AAI71194 NP_777287 NP_001121984 ABP96714 CAC06714

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Table. 1 Species used in this study.

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CGCCGTGGACATTACAT TCCACATCAGACCCACC TTGAGCCAGAACCATTTGC CAGGCAAGACGGCTAAG

cDNA fragment PCR cDNA fragment PCR 5'-RACE 3'-RACE

TCATTCATCACCATAGCCAACA CTCGCAAGACCAAACTCCATAA ACCATTTACAGAAGCCTCCA TCTCCACATCAGACCCACCA GGTGTGGCTATGAATCCTGT

qPCR qPCR qPCR qPCR qPCR (control)

ACGACGAGCAGCAATAAGAC

qPCR (control)

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RPL8R

Purpose

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cDNA cloning primers ESR2F ESR2R 5'-GSP1 3'-GSP1 Gene expression primers ESR1L ESR1H ESR2L ESR2H RPL8F

Sequence (5ꞌ→3ꞌ)

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Primer

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This study This study This study

Zhang et al., 2016 This study Katsu et al., 2004

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Geospiza fortis Zonotrichia albicollis Sturnus vulgaris Columba livia Alligator sinensis Alligator mississippiensis Pelodiscus sinensis Chelonia mydas Pseudemys nelsoni Anolis carolinensis Bradypodion pumilum Plestiodon finitimus Elaphe quadrivirgata 100 Protobothrops flavoviridis Homo sapiens Mus musculus Bos grunniens Sus scrofa Xenopus laevis 100 Xenopus tropicalis Danio rerio Oryzias latipes Lepomis macrochirus Oncorhynchus mykiss A. sinensis (ESR1) 96

63 100 92

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The complete cDNA of Chinese alligator ESR2 was cloned for the first time. Phylogenetic tree support the position of crocodilians as the sister group of birds. 3D model of Chinese alligator ESR2 with estradiol was constructed.

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Sex-specific tissue gene expression of ESR1 and ESR2 are obvious.

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ESRs expression change significantly in the pituitary during the reproductive cycle.

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