Follicle-stimulating hormone receptor (FSHR) in Chinese alligator, Alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the female reproductive cycle

Animal Reproduction Science 156 (2015) 40–50

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Follicle-stimulating hormone receptor (FSHR) in Chinese alligator, Alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the female reproductive cycle Rui Zhang a , Shengzhou Zhang a,∗ , Xue Zhu a , Yongkang Zhou b , Xiaobing Wu a,∗ a

Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China b Alligator Research Center of Anhui Province, Xuanzhou 242000, China

a r t i c l e

i n f o

Article history: Received 28 November 2014 Received in revised form 6 February 2015 Accepted 20 February 2015 Available online 28 February 2015 Keywords: Chinese alligator FSHR cDNA cloning Homology analysis Gene expression

a b s t r a c t The follicle-stimulating hormone (FSH) plays a central role in vertebrate reproduction, with the actions of FSH mediated by FSH receptors (FSHRs) on the granulosa cells of the ovary. The present study reports the cloning and characterization of FSHR in Chinese alligator, Alligator sinensis (caFSHR), and its tissue distribution and mRNA expression changes during the reproductive cycle. The mature protein of caFSHR displays typical features of the glycoprotein hormone receptor family, but also contains some remarkable differences when compared with other vertebrate FSHRs. The deduced amino acid sequence of the caFSHR shares identity of 85% with Chinese softshell turtle, 84–87% with birds, 77–78% with mammals, 67–73% with amphibians and 51–58% with fishes. Phylogenetic tree analysis of the FSHR amino acid sequence indicated that alligators cluster into the bird branch. Tissue expression analysis showed that caFSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid. Expression of caFSHR in the ovary is high in May (breeding prophase) and peaks in July during the breeding period, where it is maintained at high levels through September (breeding anaphase). Expression decreases significantly in November (hibernating period) and then remains relatively low from January to March (hibernating period). These temporal changes in FSHR expression suggest that it plays an important role in promoting ovarian development during the female reproductive cycle of Chinese alligator. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The Chinese alligator, Alligator sinensis, belonging to the Crocodilia Alligatoridae family, is an endangered species

∗ Corresponding authors. Tel.: +86 139 55370129; fax: +86 553 3836873. E-mail addresses: [email protected] (S. Zhang), [email protected] (X. Wu). http://dx.doi.org/10.1016/j.anireprosci.2015.02.008 0378-4320/© 2015 Elsevier B.V. All rights reserved.

indigenous to China and was listed by the Chinese government as a first-level state-protected species in 1972 (Yan et al., 2005). In response, nature reserves and artificial farms of the Chinese alligator were set up in Anhui and Zhejiang Provinces. Though the issues with artificial incubation and breeding of the Chinese alligators have been successfully resolved, the productivity of the Chinese alligator is considerably affected by inherent problems such as lower and highly annual fluctuating egg-laying rate (Cheng et al.,

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2003). The cause of these problems remains unclear. Application of assisted reproductive technologies (e.g. hormonal treatment) has the potential to improve reproductivity. The reproduction of vertebrates is mainly under the endocrine control of hypothalamus–pituitary–gonad axis. The pituitary gonadotropins, follicle-stimulating hormone (FSH), a glycoprotein hormone, is the central hormone regulating vertebrate reproduction, it functions together with luteinizing hormone(LH) to promote the growth and development of gonads, to control gametogenesis and regulate gonadal endocrine functions (Chauvigné et al., 2012). In females, FSH stimulates ovarian follicular development and egg production (Zhao et al., 2010). The follicles receiving insufficient FSH support are doomed to atretic degeneration during the critical stage of follicle development (Zhao et al., 2010). FSH could increase serum oestradiol17 beta concentrations, number of growing follicles and yolk deposition in aging hens (Gallus gallus domesticus) with decreased egg production (Palmer and Bahr, 1992). Expression of FSH␤ in the Chinese alligator ovary increases significantly during the breeding prophase and reaches a peak in the breeding period, then decreases significantly during the breeding anaphase and hibernating periods (Zhang et al., 2015). In vivo FSH challenge in five-monthold female American alligators (Alligator mississippiensis) could increase circulating estradiol concentrations, ovarian steroidogenic enzyme mRNA expression levels, and oviducal mRNA expression levels of sex steroid hormone receptors, activin-binding proteins, and mitotic factors (Moore et al., 2012). FSH exerts its biological activities through interaction with specific receptors (FSHRs) present on target cell surfaces (Minj et al., 2008). FSHR belongs to the large family of G protein-coupled receptors (GPCRs), which also includes the TSH receptor (TSHR) and the LH receptor (LHR). FSHR, together with TSHR and LHR, constitute the subfamily of glycoprotein hormone receptors (GpHRs) that are a part of a broader family of leucine-rich repeat-containing GPCRs (Szkudlinski et al., 2002). Members of this family are characterized by a large extracellular (EC) domain with multiple imperfect leucine-rich repeats (LRRs), flanked by N- and C-terminal cysteine-rich sub-domains, and a rhodopsin-like domain of seven transmembrane (7TM) helices followed by a C-terminal intracellular tail (Hsu et al., 2000). Residues present in the ␤-strand motifs of the LRRs provide specificity and affinity for ligand binding, while the TM domain is responsible for receptor activation and signal transduction through G proteins (Smits et al., 2003). Hormone binding to the EC domain of a GpHR induces changes in the 7TM domain that promote cAMP accumulation via activation of heterotrimeric Gs, which then initiate a signaling cascade leading to steroid synthesis (Szkudlinski et al., 2002). The nucleotide and amino acid sequences of FSHRs have been extensively studied in mammals, birds and fish, wherein conservation of the molecular structure of FSHRs was found, while some specific features were also reported in different species (Kumar et al., 2001; Zhao et al., 2010). Relatively less data are available concerning the molecular structure of reptilian FSHRs. In Crocodilia, only two partial FSHR mRNA sequences for the American alligator (DQ010157.1 and JN568480.1) and two

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predicted FSHR mRNA variants for the Chinese alligator (XM 006014720.1 and XM 006014721.1) are available in GenBank, no Crocodilian FSHRs have yet been characterized. FSHR was found to be highly expressed in granulosa cells of the ovary and in Sertoli cells of the testis (Rocha et al., 2007; An et al., 2009; Shen and Yu, 2002). However, many studies on bird and fish tissue show that FSHR is also expressed in non-gonadal tissues (Zhao et al., 2010; Mu et al., 2013). For example, in chick, FSHR was also expressed in the pancreas and glandular stomach (Zhao et al., 2010); in Korean rockfish, FSHR was also detected in the spleen, head kidney, caeca, heart, liver, gill, kidney and intestine (Mu et al., 2013); and in channel catfish, FSHR was also expressed in the spleen (Kumar et al., 2001). These results suggest that FSHR expression is not restricted to gonadal tissues and may have various physiological functions in different tissues. To assess its roles in regulating ovarian development, the temporal changes in FSHR expression have been documented in the gonadal tissues of various vertebrate species during sexual maturation, including mammals, birds and fish, such as mouse (O’Shaughnessy et al., 1996), chick (Zhao et al., 2010), Zi geese (Kang et al., 2010), Korean rockfish (Mu et al., 2013), channel catfish (Kumar et al., 2001), whereas no related work has been reported in the Crocodilian order so far. In the present study, we report the molecular cloning and characterization of FSHR in the Chinese alligator, and its tissue distribution and mRNA expression changes during the female reproductive cycle. The aim of this study was to expand our knowledge of FSHR phylogenetic evolution and to provide basic data for further study on the mechanisms that regulate Chinese alligator reproduction, which could be useful for maximizing the success of artificial breeding. 2. Materials and methods 2.1. Animals and RNA isolation Sexually mature female Chinese alligators were obtained from the Xuanzhou Alligator Culturing Centre of Anhui Province. Ovary, stomach, intestine, pancreas, liver, heart, thymus, thyroid and oviduct tissues were excised and immediately put into RNA-Be-Locker A (Sangon Biotec, Shanghai) and stored at −80 ◦ C. Tissue (100 mg) was ground in liquid nitrogen, followed by extraction of total RNA using Total RNA Extractor (Sangon Biotec, Shanghai) according to the manufacturer’s instructions. To remove genomic DNA contamination, RNase-free DNase I (TaKaRa, Dalian, China) was used. Concentration of total RNA was determined by measuring absorbance at 260 nm, and purity was determined by dividing absorbance at 260 nm by absorbance at 280 nm (A260 /A280 ). Only RNA samples with A260 /A280 ratios of 1.8–2.0 were used for RT-PCR. Total RNA was stored at −80 ◦ C until further use. 2.2. Cloning of the coding region of caFSHR cDNA Reverse transcription of mRNA into cDNA was achieved using PrimeScript 1st cDNA Synthesis Kit (TaKaRa, Dalian).

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Table 1 Primers used for cloning cDNA of the Chinese alligator FSHR. Primer names

Primer sequences

Technique

FH1S FH1R 5 -GSP1 5 -GSP2 5 -GSP3 3 -GSP1 3 -GSP2 YFH2S YFH2R ␤-act1S ␤-act1R

5 -CCTCGATTTCTAATGTGC-3 5 -GAGATGACATTGCGGTTT-3 5 -TGCACCTGTTTGCCAA-3 5 -TGGCTTTTGGTCTTTATGTCC-3 5 -CAGCAGCAGATAGATGCCT-3 5 -TGCTTTCACTGTAGCCCTTCTTCCCAT-3 5 -GCCTGCCAATGGATATAGACATGCCCT-3 5 -ACGGTGTTTGCGAGTGAA-3 5 -ATGGGAAGAAGGGCTACA-3 5 -ACCGAAACAAGAACCCAT-3 5 -CCGACACGCTAAGACTGC-3

RT-PCR RT-PCR 5 RACE 5 RACE 5 RACE 3 RACE 3 RACE qRT-PCR qRT-PCR qRT-PCR qRT-PCR

Primers used are shown in Table 1 and were designed around the most highly conserved regions of the FSHR nucleotide sequence from three different species (Chinese softshell turtle, mallard and chicken). The conserved sequence of FSHR cDNA was obtained from NCBI’s GenBank. Total cDNA from the ovaries of Chinese alligators was used as a template for subsequent polymerase chain reactions (PCRs), and a partial read of the FSHR cDNA was obtained using primer pair FH1S and FH1R. Another set of primers was designed to obtain other upstream and downstream regions of the FSHR sequence by using RACE (rapid amplification of cDNA end methods) (Table 1). Primers 5 GSP1, 5 -GSP2 and 5 -GSP3 were used to obtain the 5 RACE product, and primers 3 -GSP1 and 3 -GSP2 were used to obtain the 3 RACE product. All primers used in this study were designed using PrimerPrimer 5.0 and synthesized by Sangon Biotec (Shanghai). The 5 RACE and 3 RACE reactions were performed using the System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, USA) and SMARTerTM RACE cDNA Amplification Kit (Clontech, USA), according to the manufacturers’ instructions. PCR products were purified using a DNA Gel Extraction Kit (Axygen, Hangzhou), and purification products were sub-cloned into PMD-18 T (TaKaRa, Dalian) followed by transformation into DH5␣ E. coli cells (TransGen, Beijing). Nine to ten positive clones were randomly selected for DNA sequencing by Sangon Biotech (Shanghai). 2.3. cDNA sequence analysis of caFSHR Amplified PCR products were confirmed to be FSHR based on high homology with FSHR sequences from other species, according to the NCBI’s Blast tool. The partial cDNA of the Chinese alligator FSHR sequence was aligned and compared with FSHR of a few representative species using ClustalX. Amino acids were deduced using Protein Translation, a web-based program at ExPaSy (http://web.expasy.org/translate/). The ORF was found using ORF finder on the NCBI website (http://www.ncbi.nlm.nih.gov./projects/gorf/). The signal peptide was predicted by the SignalP 4.1 server (http://www.cbs.cbs.dtu.dk/services/SignalP/). Potential N-glycosylation sites were predicted using the tool at http://www.cbs.dtu.dk/services/YinOYang/. Prediction of Ser, Thr, and Tyr phosphorylation sites was done at

http://www.cbs.dtu.dk/services/NetPhos/. The presence and location of the putative signal peptide cleavage site, the 7TM helices and potential N-glycolylation sites in the amino acid sequences were predicted using the prediction servers of the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/). Multiple amino acids sequences of different species were aligned using ClustalX.

2.4. Tissue specificity of FSHR gene expression To examine the tissue specificity of FSHR expression, total RNA was isolated from various tissues as described above, including ovary, stomach, intestine, pancreas, liver, heart, thymus, thyroid and oviduct tissues. Total RNA from each tissue was reverse transcribed to cDNA, and then subjected to PCR amplification (31 cycles) of the coding region of the caFSHR cDNA using the primer pair FH1S and FH1R. The cDNA of each tissue was also subjected to PCR amplification (31 cycles) of the Chinese alligator ␤-actin gene as an internal reference. PCR products were detected using 1.5% agarose gel electrophoresis.

2.5. Reproductive cycle changes in FSHR gene expression To investigate the female reproductive cycle changes in FSHR gene expression, samples from ovaries were collected in January, March, May, July, September and November. Total RNA was isolated as described above. The same amount of total RNA (100 ng) from each sample was reverse transcribed to cDNA and then subjected to realtime quantitative-PCR (qRT-PCR) analysis to examine the expression level of caFSHR. Each 20 ␮l PCR mixture contained 10 ␮l of 2× iQTM SYBR Ex TaqTM (Tli RNaseH Plus), 0.4 ␮l (100 mM) of each primer, and 0.8 ␮l of cDNA, with ddH2 O accounting for the remaining volume. Mixtures were incubated in an iCycler iQ Real-time Detection system (Bio-Rad, Hercules, CA), and the qRT-PCR program ran as follows: after an initial denaturation step at 95 ◦ C for 30 s, 40 cycles were carried out at 95 ◦ C for 30 s, followed by annealing at 60 ◦ C for 30 s. A melting curve was constructed to verify that only a single PCR product was amplified. Samples were assayed in triplicate with standard deviations of threshold cycle values not exceeding 1 on a within-run basis. Relative expression was normalized using the internal reference gene ␤-actin and quantified using the formula 2−Ct . Statistical differences between groups were determined by one-way ANOVA followed by the Tukey multiple comparison test.

2.6. Construction of a phylogenetic tree of vertebrate FSHR A phylogenetic tree of selected vertebrate FSHRs was constructed using the neighbor-joining method based on the aligned amino acid sequences (Molecular Evolutionary Genetic Analysis, MEGA 6.0). To derive the confidence value for this analysis, bootstrap trials were replicated 1000 times.

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3. Results 3.1. Amplification of the full-length caFSHR cDNA RNA gel electrophoresis showed two distinct bands of 18s and 28s in the RNA products, suggesting sufficient quality for follow-up experiments. Total RNA was reverse transcribed to synthesize cDNA, which was used as a template for PCR amplification with primer pair FH1S and FH1R (Table 1). Agarose gel electrophoresis showed a distinct band at the size of the anticipated fragment (Fig. 1a), which yielded a 500-bp cDNA fragment after sequencing. Primers 5 -GSP1, 5 -GSP2 and 5 -GSP3 (Table 1) were used to obtain the 5 RACE product (Fig. 1b), a 1453-bp fragment that was then sequenced. Primers 3 -GSP1 and 3 -GSP2 (Table 1) were used to obtain the 3 RACE product (Fig. 1c), which was a DNA fragment of 1010-bp after sequencing. Results obtained using NCBI’s BLAST tool to align the sequences indicated that they were all partial fragments of the FSHR cDNA. 3.2. Sequencing and identification of the cDNA

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of LRR1–3 and LRR5–8 found to be identical with those in FSHRs of mammals, birds, amphibians and Chinese softshell turtle, while those of LRR4 and LRR9 having two different residues (L128 and A257) than those in FSHRs of the same species. The 7TM sequences of the caFSHR were also found to have high identity with those of the other vertebrates previously mentioned. Only six amino acids were different between caFSHR and Chinese softshell turtle FSHR (488 A, 489 A, 497 L, 507 I at TMIV and 533 V, 545 V at TMV). In addition, only nine amino acids were different between caFSHR and chicken FSHR, with the most variable regions located in TMIV of these FSHRs. The conserved ERW motif is found at the bottom of the predicted TM III domain and contains Arg469 and Glu468 , which form an ionic lock with Asp569 at the cytoplasmic end of TM VI. The NPxxY motif is also found in the TM VII domain of caFSHR and includes Asn633 , one of the most conserved residues in rhodopsin-like GPCRs. Also, one potential serine phosphorylation site exists in the third intracellular loop at position 566, and eight potential phosphorylation sites (662 S, 663 S, 663 T, 660 T, 675 T, 675 T, 630 Y, and 674 Y, 674 Y) exist in the carboxyterminal region.

The 500-bp cDNA fragment and 5 - and 3 RACE fragments were assembled using DNASTAR software. Alignment yielded a total sequence length of 2643-bp (Fig. 2) consisting of 78-bp of the 5 -untranslated region (UTR), 2088-bp of the open reading frame and 477-bp of the 3 -UTR. One canonical polyadenylation signal (AATAAA) was found 42-bp upstream of the poly(A) tail, and one “adenylate + uridylate rich (AU-rich)” AUUUA motif was found within the 3 -UTR, which is a signal for rapid mRNA degradation. The deduced amino acid sequence of caFSHR is shown in Fig. 2. The coding sequence of caFSHR is a 2088bp ORF encoding a protein of 695 amino acids, of which the first 17 comprise the signal peptide. In terms of receptor structure, the extracellular Nterminal domain of caFSHR is 368 amino acids long. The transmembrane region is formed by 262 amino acids organized into seven transmembrane-spanning domains, and the cytoplasmic tail is formed by 65 amino acids. Five putative N-linked glycosylation sites of caFSHR were found on Asn at positions 47, 191, 199, 268 and 293. Pfam database searches and further comparison with human FSHR allowed the identification of nine imperfect LRRs in the caFSHR. These LRRs are flanked by ten conserved cysteines, four of which are in an N-terminal cluster (Cys at positions 19, 24, 26 and 33) and the rest found within a C-terminal group (Cys at positions 275, 276, 292, 340, 348 and 358). Finally, one Protein Kinase C phosphorylation site was found in the cytoplasmic end of TM VI at position 634.

3.4. Homologous and phylogenetic analysis

3.3. Amino acid sequence alignment

The Chinese alligators exhibit a distinct seasonal reproductive cycle, including breeding prophase (March to May), breeding period (June to August), breeding anaphase (September to October) and hibernating period (November to next February). The ovarian follicles are smaller in size of 5–20 mm during hibernating period, they grow rapidly and attain 20–30 mm in diameter in late breeding prophase, their size continues to increase and ovulation occurs once follicles reach a diameter of 30–40 mm during breeding

The amino acid sequence of caFSHR was compared to that of mammals, birds, amphibians, fish and other reptiles (Fig. 3), and four highly conserved GpHR signature sequences (e.g. 271 YPSHCCAF, 355 FNPCEDIMG, 468 ERW, 581 FTD, 624 NPFLY) were found. The aminoterminal exodomain of the caFSHR has an LRR sub-domain with nine leucine-rich repeats, with the amino acid sequences

Alignment of FSHR nucleotide and amino acid sequences between the Chinese alligator and other selected vertebrates indicates that the deduced amino acid sequence of the caFSHR shares identity of 85% with Chinese softshell turtle, 84–87% with birds, 77–78% with mammals, 67–73% with amphibians and 51–58% with fishes (Table 2). The phylogenetic tree of these FSHR amino acid sequences also shows that species in the same animal classes are clustered in groups, with the Chinese alligator and Chinese softshell turtle forming a branch that then clusters with birds. This suggests that reptilian FSHRs exhibit a closer phylogenetic relationship with birds than with other mammals and amphibians (Fig. 4). 3.5. Tissue specificity of caFSHR gene expression The tissue distribution of caFSHR in the ovary, stomach, intestine, pancreas, liver, heart, thymus, thyroid and oviduct of female Chinese alligators was analyzed by RTPCR. As shown in Fig. 5, caFSHR was not only expressed in the ovary, but also in the stomach, intestine, pancreas, liver and oviduct at similar levels, while it was not detectable in heart, thymus or thyroid. 3.6. Reproductive cycle changes in caFSHR gene expression

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Fig. 1. Electrophoresis of PCR products. Note: (a) Conventional PCR product. (b) 5 RACE product. (c) 3 RACE product, M shows the DNA marker, I shows the sample band.

period. The relative content of caFSHR mRNA in the ovary during a reproductive cycle was examined by qRT-PCR. As shown in Fig. 6, caFSHR was highly expressed in May (breeding prophase) and reached a peak in July (breeding period). High expression was maintained through September (breeding anaphase), but decreased significantly in November (hibernating period). Lower levels were also maintained from January to March (hibernating period). 4. Discussion In the present study, we cloned the full-length cDNA of FSHR from the Chinese alligators and determined the

nucleotide and amino acid sequences and structures. To our knowledge, this is the first study to show the entire nucleotide and deduced amino acid sequence of FSHR from the Crocodilian order. The caFSHR was predicted to contain an EC domain (368 amino acids plus a signal peptide (from 1 to 17), a putative 7TMD (262 amino acids) and an intracellular C-terminal domain (65 amino acids). It also was found to contain all the general structural features of a glycoprotein hormone receptor (e.g. 581 FTD, 624 NPFLY) (Vassart et al., 2004), including a highly conserved amino acid sequence (YPSHCCAF) at positions 271–278 that forms a pocket for specific glycoprotein hormone binding (Lloyd and Griswold, 1995). The caFSHR possesses five potential N-glycosylation sites (47 NST, 191 NGT, 199 NLS, 268 NLT,

Table 2 Comparison of the percent identity of mature FSHR proteins between the Chinese alligator and representative vertebrate species based on nucleotide and amino acid sequences. Taxonomy

Species name

Scientific name

GenBank ID

Nucleotide identity (%)

Amino acid identity (%)

Amino residue

Mammals

Human Sheep Bovine Rat

Homo sapiens Ovis aries Bos taurus Rattus norvegicus

NM NM NM NM

000145.3 001009289.1 174061.1 199237.1

76 75 75 75

78 77 77 77

695 695 695 692

Birds

Chicken Mallard Rock pigeon Zebra finch

Gallus gallus Anas platyrhynchos Columba livia Taeniopygia guttata

NM 205079.1 XM 005012095.1 XM 005498409.1 XM 002196132.2

86 83 82 82

83 87 85 85

693 693 694 693

Reptiles

Chinese alligator Common Lancehead Chinese softshell turtle

Alligator sinensis Bothrops jararaca Pelodiscus sinensis

This study AY189696.1 XM 006122101.1

– 77 88

– 76 85

695 673 669

Amphibians

African clawed frog Toad

Xenopus Laevis Rana rugosa

NM 001256260.1 AB602925.1

72 69

73 67

692 663

Fishes

Zebrafish European eel Orange-spotted grouper Japanese medaka

Danio rerio Anguilla anguilla Epinephelus coioides Oryzias latipes

NM 001001812.1 AB700600.1 HQ650769.1 NM 001201514.1

66 69 66 66

56 58 51 51

668 660 701 687

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Fig. 2. The nucleotide and deduced amino acid sequences of Chinese alligator FSHR. Note: Upper sequence depicts amino acids, whereas the lower shows the nucleotide sequence. ATG indicates initiation codon. TGA indicates termination codon. Five putative N-linked glycosylation sites are denoted by rectangles. Cysteine residues of the N-and C-terminal cysteine-rich regions of the EC domain are indicated by asterisks. The position of the seven predicted TM helices is shown as gray boxes. Double underlines indicate Protein Kinase C phosphorylation sites. Motifs of ATTTA in the 3 -UTR are denoted by rectangles and gray. The polyadenylation signals (AATAAA) are indicated with open rectangles and black.

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Fig. 3. Comparison of FSHR amino acid sequences between Chinese alligator and other representative vertebrate species. Note: Hyphens/gaps (−) were introduced in order to obtain maximum alignment. Conserved residues are indicated by a “*” under sequences. The cysteine residues of the N-terminal are marked with single underlines. The nine ␤-strand motifs (X-L-X-L-X) of the LRRs, identified by Pfam Blast and sequence alignment with the human FSHR, are shown as rectangles and gray. The seven TM helices are shown as rectangles. Highly conserved GpHR signature motifs involved in receptor function and conformation are indicated in gray. Double underlines indicate phosphorylation sites in the carboxyterminal region.

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Fig. 4. Phylogenetic tree of FSHR amino acid sequences from Chinese alligator and other representative vertebrates. Note: The source and references of the selected FSHR data are indicated in Table 2.

293 NIS) in the aminoterminal region, compared to six and four sites found in the snake and human FSHRs (Bluhm et al., 2004; Davis et al., 1995), two of which are conserved (191 NGT and 199 NLS). The varying need for glycosylation sites is likely related to proper folding and trafficking of the receptor protein to the membrane. In addition, Thr634 in caFSHR was found to be a potential phosphorylation site for Protein Kinase C in the intracellular C-terminal domain, which is conserved in birds but differs in mammals. The aminoterminal exodomain of the caFSHR has an LRR sub-domain with nine leucine-rich repeats. The primary function of LRRs is to provide a versatile structural framework for protein–protein interaction (Kajava and Kobe, 2002). LRRs usually start with ␤-strands composed of a highly conserved X-L-X-L-X motif, in which X indicates any amino acid and L refers to leucine, isoleucine or other hydrophobic residues (Kobe and Kajava, 2001). Our study found nine LRRs in caFSHR, which is consistent with that of reptiles (Bluhm et al., 2004), birds (Zhao et al., 2010) and mammals (Gromoll et al., 1992), though differs from that in some fishes (Mu et al., 2013). The presence or absence

of LRRs could lead to changes in the shape of the hormonebinding domain, thereby altering hormone recognition, specificity or activation properties in these species. The LRRs are flanked by two cysteine-rich sub-domains with 10 cysteine residues total. Similar to mammals and birds, the N-terminal cysteine-rich region contains four cysteines (19 C, 24 C, 26 C and 33 C) capable of forming two disulfide bridges, whereas some fish (e.g. black porgy, zebrafish and european eel) only have two cysteines (An et al., 2009; Ji et al., 2013; Minegishi et al., 2012). These results suggest similarity in the folding of FSHRs within this region among alligator, birds and mammals, but not fish. In addition, the six cysteines in the C-terminal cysteine-rich subdomain were found to be conserved in Chinese alligator and all other species analyzed. The TM domain of FSHR includes seven stretches of 21–23 predominantly hydrophobic residues predicted to form ␣-helices, and connected by three intracellular and three EC loops (Rocha et al., 2007). Alignment of the caFSHR with FSHRs from several species showed some variability in regions TMIV and TMV of caFSHR, while other TM sequences were found to have high identity with those of

Fig. 5. Tissue specificity of FSHR gene expression analyzed by RT-PCR. Note: 100 ng of total RNA from ovary (lane 1), stomach (lane 2), intestine (lane 3), pancreas (lane 4), liver (lane 5), heart (lane 6), thymus (lane 7) and thyroid (lane 8) and oviduct (lane 9) was subjected to RT-PCR for FSHR cDNA and ␤-actin amplification (31 cycles), with ␤-actin serving as a reference gene. M shows the DNA marker.

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Fig. 6. Reproductive cycle changes of caFSHR gene expression in Chinese alligator ovary. Note: Each bar represents mean ± SEM of three samples. The same letters indicate that the treatments are not significantly different as determined by one-way ANOVA. Different letters indicate that the treatments are significantly different as determined by one-way ANOVA (P < 0.05).

other vertebrates. This is particularly true for the specific functional regions, i.e. the conserved ERW motif, which can form an ionic lock with Asp569 , activation of the FSHR receptor involves the disruption of this ionic lock, thus leading to movement of these two ␣-helices. The NPxxY motif, which includes Asn633 , it has been suggested that this residue would be implicated in the activation of the receptor by switching its interaction between two aspartic residues (Vassart et al., 2004). The C-terminal domain, rich in potential phosphorylation sites, is quite different in many species, which may lead to the differential regulation of GTHRs between species (Gether, 2000). Eight potential phosphorylation sites in the carboxyterminal region of the caFSHR were identified, compared to six and five of chick and human FSHRs, respectively (Zhao et al., 2010; Lazari et al., 1999). Meanwhile, FSHRs of chick and human have one and three potential phosphorylation sites, respectively, in the third intracellular loop, whereas caFSHR has only one. Phosphorylation of the gonadotropin receptors accompanies their activation, which affects uncoupling of the receptor from the G-protein and leads to receptor internalization (Lazari et al., 1999). The intracellular loops have been characterized as the region of phosphorylation in the rat FSHR (Nakamura et al., 1998), thus it seems likely that the mechanism of regulation of the caFSHR is identical with that of birds, but differs from that of the mammals. Two cysteine residues at positions 644 and 645 are conserved in birds and caFSHR, and have been found to be involved in receptor coupling and regulation of signal transduction (Simoni et al., 1997), whereas the mammalian FSHRs possess one cysteine each at positions 644 and 672. Overall, we found many structural similarities in the FSHRs of Chinese alligator and birds. As anticipated, the homology of the cloned caFSHR with Mallard FSHR was very high (83% and 87% for nucleotide and amino acid sequences, respectively) whereas as a lower degree of similarity was found between zebrafish (66% and 56%, respectively) and European eel FSHR (69% and 58%, respectively). Meanwhile, phylogenetic analysis of the amino acid sequences indicated that caFSHR is clustered into the bird FSHR branch, which is in accordance with the phylogeny of TSHR (Helbing et al., 2006), suggesting that a close evolutionary relationship exists between alligators and birds, supporting the hypothesis that alligators are a sister group to birds.

The tissue distribution of caFSHR expression was analyzed by RT-PCR and expression was found not only in the ovary, but also in the stomach, intestine, pancreas and liver (though not in the heart, thymus or thyroid). This differs from previous reports showing expression of FSHR only in granulosa cells of the ovary and Sertoli cells of the testis (Rocha et al., 2007; An et al., 2009). Our results are supported by other studies showing that FSHR has also been detected in almost all types of non-gonadal tissues from various species, including heart, brain, liver, spleen, lung, kidney, pancreas, rumen, duodenum, muscle, fat, hypothalamus, pituitary and oviduct (Pan et al., 2014; Zhao et al., 2010; Mu et al., 2013; Kumar et al., 2001; Moore et al., 2012). The distribution of FSHR in non-gonadal tissues may be a common biological phenomenon, though the distribution pattern varies between species. The specific biological actions of FSHR in the extra-gonadal tissues remain unclear (Pakarainen et al., 2007), it will surely be an interesting issue to address in the future. The role of FSHR in the regulation of ovarian development has been studied in several species by assessing the temporal expression changes of FSHR in gonadal tissue during oocyte maturation. Mu et al. (2013) have demonstrated that Korean rockfish FSHR transcript abundance increases with oocyte growth, and higher abundance is seen during early stages compared with late stages. However, in channel catfish, FSHR expression was low during the whole reproductive cycle but suddenly increased after ovulation, which differed from that of most fish (Hirai et al., 2000). In Zi geese, the level of ovarian FSHR mRNA increased significantly from the postnatal to egg-laying stage, with the exception of months 2 and 3 (Kang et al., 2010). A similar expression pattern was reported in chickens (Zhao et al., 2010). There were yet no reports regarding the expression changes of FSHRs in reptiles during the reproductive cycle. In the present study, we found that caFSHR expression in the Chinese alligator ovary increased significantly during the breeding prophase and reached a peak in the breeding period, then decreased significantly during the breeding anaphase. These results are similar to the variation in FSHR expression observed in Zi geese and chicken, but different from that of channel catfish. Interestingly, the temporal expression of caFSHR in the ovary of Chinese alligator is in coincidence with that of FSH␤ (Zhang et al., 2015), which implicated that FSH/FSHR might play an important role in promoting ovarian development during the reproductive cycle. In summary, the complete cDNA sequence of caFSHR was determined for the first time in this study, and changes in spatial and temporal expression of caFSHR during the female reproductive cycle were investigated by RT-PCR and qRT-PCR. The sequence of caFSHR exhibits a higher degree of amino acid identity with birds than other species, and caFSHR was expressed not only in the ovary, but also in the stomach, intestine, pancreas, liver and oviduct, though not in heart, thymus or thyroid. Whether this special distribution has additional effects on the stomach, intestine, pancreas, liver and oviduct are unknown at this time. With respect to its impact on the reproductive cycle, caFSHR is found to be highly expressed in May (breeding prophase) and reaches a peak in July (breeding period), where it

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is maintained at a high level through September (breeding anaphase) and decreases significantly in November (hibernating period). Relative expression of caFSHR is low from January to March (hibernating period). These temporal changes in FSHR expression indicate that it may play an important role in promoting ovarian development during the reproductive cycle. In addition, knowledge of the structure of caFSHR obtained from this study may aid further studies related to structure–function relationships. Finally, our studies found that the hormone-binding domain of caFSHR has high similarity in structure with those of mammalian and bird FSHRs. Based on this information, it may be possible to increase FSH levels in Chinese alligator by using mammalian and bird FSH, which could stimulate the ovary to produce more estrogen and improve the reproductivity. Conflict of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Sources of support This work was sponsored by Natural Science Foundation of Anhui Province (Grant No. 11040606M75) and National Natural Science Foundation of China (NSFC, Grant No. 31272337). References An, K.W., Lee, K.Y., Yun, S.G., Choi, C.Y., 2009. Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy. Acanthopagrus schlegeli: effects of estradiol-17␤ on mRNA expression profiles. Comp. Biochem. Physiol. B 152, 177–178. Bluhm, A.P., Toledo, R.A., Mesquita, F.M., Pimenta, M.T., Fernandes, F.M., Ribela, M.T., Lazari, M.F., 2004. Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor. Gen. Comp. Endocrinol. 137, 300–311. Chauvigné, F., Verdura, S., Mazón, M.J., Duncan, N., Zanuy, S., Gómez, A., Cerdà, J., 2012. Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost. Biol. Reprod. 87, 35. Cheng, B.H., Hua, T.M., Wu, X.B., 2003. Research on the Chinese Alligator. Shanghai Scientific and Technical Publishers, Shanghai (in Chinese). Davis, D., Liu, X., Segaloff, D.L., 1995. Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function. Mol. Endocrinol. 9, 159–170. Gether, U., 2000. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev. 21, 90–113. Gromoll, J., Gudermann, T., Nieschlag, E., 1992. Molecular cloning of a truncated isoform of the human follicle stimulating hormone receptor. Biochem. Biophys. Res. Commun. 188, 1077–1083. Helbing, C.C., Crump, K., Bailey, C.M., Kohno, S., Veldhoen, N., Bryan, T., Bermudez, D., Guillette Jr., L.J., 2006. Isolation of the alligator (Alligator mississippiensis) thyroid hormone receptor alpha and beta transcripts and their responsiveness to thyroid stimulating hormone. Gen. Comp. Endocrinol. 149, 141–150. Hirai, T., Oba, Z.X., Yao, Z.X., Chang, X.T., Yoshiura, Y., Kobayashi, T., Nagahama, Y., 2000. Putative gonadotropin receptors in tilapia (Oreochromis niloticus) gonads: cDNA cloning and expression during oogenesis. In: Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E., Stefansson, S.O. (Eds.), Proceedings of the Sixth International

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