Molecular cloning of estrogen receptor α of the Nile crocodile

Comparative Biochemistry and Physiology, Part A 143 (2006) 340 – 346 www.elsevier.com/locate/cbpa

Molecular cloning of estrogen receptor α of the Nile crocodile Yoshinao Katsu a,b , Jan Myburgh c , Satomi Kohno d , Gerry E. Swan c , Louis J. Guillette Jr. d , Taisen Iguchi a,b,⁎ a

b

Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan Department of Molecular Biomechanics, School of Life Sciences, Graduate University of Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan c Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag 04, Onderstepoort 0110, Republic of South Africa d Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611, USA Received 24 April 2005; received in revised form 6 December 2005; accepted 7 December 2005

Abstract Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor α (ERα) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERα cDNA was obtained using 5′ and 3′ rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERα showed high identity to the American alligator ERα (98%), caiman ER (98%), lizard ER (82%) and chicken ERα (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERα was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERα in crocodiles and expands our knowledge of estrogen receptor evolution. © 2005 Elsevier Inc. All rights reserved. Keywords: Crocodile; Estrogen receptor α; Cloning; Ovary; Evolution; Reptile

1. Introduction Estrogens play important roles in the reproductive biology of vertebrates including reptiles. In female reptiles, estrogens exhibit a pronounced seasonal cycle coincident with reproductive activity (for review, see Guillette and Milnes, 2001; Licht, 1984), as they induce hepatic vitellogenesis, essential for oocyte development and influence the development of the female reproductive tract stimulating hyperplasia and protein synthesis. Also, estrogens appear to play an important role in the differentiation of the ovary. The sex of all crocodilians examined to date is determined by the temperature of the nest during a ⁎ Corresponding author. Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Tel.: +81 564 59 5235; fax: +81 564 59 5236. E-mail address: [email protected] (T. Iguchi). 1095-6433/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2005.12.010

narrow window of embryonic development (Lang and Andrews, 1994; Morrish and Sinclair, 2002) rather than by sex determining genes as in mammals and birds. For the American alligator (Alligator mississippiensis), eggs incubated at 33 °C produce all male offspring whereas eggs incubated at 30 °C produce all female offspring (Lang and Andrews, 1994). A similar pattern has been described for the Nile crocodile (Hutton, 1987). Estrogens appear to play a central role in the determination of sex in those fish, birds, crocodilians and turtles with temperature sex determination (TSD) (Crews, 1996; Devlin and Nagahama, 2002). This conclusion is based, in part, on the observation that treating embryos with an estrogenic substance during the temperature sensitive period (TSP) produces female offspring, even if eggs are incubated at male producing incubation temperatures (Bull et al., 1988). Gonadal aromatase expression is elevated after differentiation of the ovary in alligators and turtles with TSD (Gabriel et al., 2001; Murdock and Wibbels, 2003; Katsu et al., unpubl. data). Estrogen receptors (ER) are

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present prior to sex determination in the genital ridge of the developing red-eared slider turtle, Trachemys scripta (Bergeron et al., 1998). ERs are present at a higher density in tissue destined to become an ovary than in tissues destined to become testis (Bergeron et al., 1998). Estrogens clearly influence ovarian differentiation; however, the precise mechanisms of estrogen action during sex determination in the reptiles are still unclear. Similar studies examining genital ridge ER expression in crocodilian embryos during the TSP have not been preformed to data, in large part because of a lack of information in the molecular biology of ER in reptiles. In vertebrates, the ERs belong to a superfamily of nuclear transcription factors that include all other steroid hormone receptors such as progestogens, androgens, glucocorticoids, mineralocorticoids, the vitamin D receptor, and the retinoic acid receptor (Blumberg and Evans, 1998). Three distinct types of ER have been isolated, to date, in vertebrates. Teleostean fish have ERα, ERβ and ERγ but the teleost ERγ form appears to be closely related to teleost ERβ suggesting it reflects a gene duplication that occurred within the teleosts (see Hawkins et al., 2000). Thus, the ancestral condition for the jawed vertebrates (Gnathostomata) appears to have been the presence of two forms of ER, corresponding to ERα and ERβ (Thornton, 2001). Indeed, these two forms of ER have been previously found in mammals, fish, birds, reptiles and amphibians. ERα sequences have been reported in reptiles, with partial sequences from two turtles; T. scripta: (Bergeron et al., 1998) and Chrysemys picta: (Custodia-Lora and Callard, 2002b), and a lizard, Anolis carolinensis, (Young et al., 1995) and full-length ERα sequences from a caiman (Caimen crocodilus) and a lizard (Cnemidophorus uniparens) (Sumida et al., 2001). Recently, ERα and ERβ were cloned and sequenced from the American alligator (A. mississippiensis) by our group (Katsu et al., 2004). To better understand the evolution of ERs in the reptiles, we isolated and sequenced ERα from ovarian tissue obtained from the Nile crocodile (Crocodylus niloticus) and examined ERα expression in the gonad. Further, we analyzed its phylogenic relationship to other vertebrate ERs. 2. Materials and methods 2.1. Animals Tissues from juvenile Nile crocodiles (C. niloticus) were collected under permit from the Thaba Kwena Crocodile Farm, Republic of South Africa on March 29, 2004 (kindly supplied by Mr. Albert Pretorius). Tissues were obtained from 1.5 m animals culled from the farm population as part of farming activities. Animals were necropsied immediately after death and small pieces of gonad and liver were obtained and placed in RNA later (Ambion, Austin, TX, USA) for later RNA isolation as described below. 2.2. Molecular cloning of estrogen receptor For the estrogen receptor (ER), two conserved amino acid regions in the DNA binding domain (MCPATN) and the ligand-

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binding domain (KCVEGM) of the ER were selected and their degenerate oligonucleotides were used as primers for polymerase chain reaction (PCR): ER-1, 5′-ATGTG(CT)CCNGCNACNAA(CT)-3′ ER-2, 5′-CATNCC(CT)TCNAC(GA)CA(CT)TT-3′. Total RNA was isolated with an RNeasy kit (Qiagen, Valencia, CA, USA). As a template for PCR, first-strand cDNA was synthesized from 2 μg of total RNA isolated from one crocodile ovary. The amplified DNA fragments were subcloned with TA-cloning plasmids pGEM-T Easy (Promega, Madison, WI), and sequenced using a BigDye Terminator Cycle Sequencing-kit (Applied Biosystems, Foster City, CA, USA) with T7 and SP6 primers, and analyzed on the ABI PRISM 377 automatic sequencer (Applied Biosystems). The 5′- and 3′-end of the ERα cDNA was amplified by rapid amplification of cDNA ends (RACE) using a SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA, USA). 2.3. Phylogenic analysis of estrogen receptor sequences All sequences generated were searched for similarity using blastn and blastp at web servers of the National Center of Biotechnology Information. Deduced amino acid sequences were aligned by using the Clustal X computer program (Jeanmougin et al., 1998), and D-domain and ligand-binding domain of their sequence were used in following analysis. The phylogenic tree was constructed using the Phylip computer program (Felsenstein, 2004) with the JTT matrix, neighbor-joining method and bootstrap resampling for 1000 times. Accession numbers of the ERα sequences included are: AAC69548 (Ictalurus punctatus), P50241 (Oryzias latipes), P50242 (Salmo salar), AAG16713 (Micropogonias undulatus), AAD31032 (Sparus aurata), O42132 (Pagrus major), AAD00245 (Oreochromis niloticus), P50240 (Oreochromis aureus), AAQ84782 (Xenopus laevis), AAQ84780 (Xenopus tropicalis), BAB79436 (Caiman crocodilus), BAD08348 (A. mississippiensis), AAB37705 (T. scripta), AAB1108 (Taeniopygia guttata), NP_990514 (Gallus gallus), AAN63674 (Coturnix japonica), BAB79437 (C. uniparens), Q9YHT3 (A. carolinensis), P03372 (Homo sapiens), Q29040 (Sus scrofa), P19785 (Mus musculus), and P06211 (Rattus norvegicus). Accession numbers of the ERβ sequences included are: AAK57823 (Squalus acanthias), AAD26921 (Carassius auratus), AAD00246 (O. niloticus), AAD31033 (S. aurata), (AAG16712 (Micropogonias undulates γ), AAG16711 (M. undulates β), BAA19851 (Anguilla japonica), Q9PTU5 (G. gallus), AAC36463 (C. japonica), Q9PVE2 (Sturnus vulgaris), BAD08349 (A. mississippiensis), AAC52602 (R. norvegicus), AAB51132 (M. musculus), BAA24953 (H. sapiens), AAD24432 (Bos taurus), and Q9XSW2 (S. scrofa). 2.4. RNA isolation and RT-PCR Total RNA was isolated with RNeasy kit (QIAGEN, Chatsworth, CA). For RT-PCR, 2 μg of total RNA was reverse transcribed using oligo (dT) primer. The following primer set was used for PCR (5′-TCCGAAAAGACCGGAGAGGTGG-3′

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and 5′-GGCACACAAATTCCTCCCCCTG-3′ for ERα). Twenty eight cycles of amplification were carried out under the following conditions: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extention at 72 °C for 1 min. The PCR products were resolved on 1.5% agarose gels. 3. Results 3.1. Cloning and sequence of crocodile ERα Using PCR techniques, partial DNA fragments were amplified from Nile crocodile ovarian RNA. A DNA fragment was obtained and sequence analysis showed that the fragment had similarity to ERα (Fig. 1). Using the RACE technique, we were able to clone the full-length Nile crocodile ERα cDNA in the 5′ and 3′ directions including the ATG start site and the TGA termination signal (Fig. 1: GenBank Accession No. AB209933). The cDNA for ERα is composed of 1764 bases and predicts a protein of 587 amino acids (Fig. 1). Comparison of the amino acid sequence of crocodile ERα with those of other vertebrates indicates that the Nile crocodile sequence is very similar to that of the American alligator (98%), caiman (98%), chicken (92%) and whiptail (Cnemidophorus) lizard (82%) (Fig. 2). Using the nomenclature of Krust et al. (1986), the Nile crocodile ERα sequence can be divided into five domains based on its sequence homology to other steroid hormone receptors. The five ERα (crocodile, alligator, caiman,

lizard and chicken) sequences examined here shared 97–77% identity in the A/B domain, 100% in the C domain (DNAbinding domain), 99–79% in the D domain, 100–91% in the E domain (the ligand-binding domain), and 100–45% in the F domain (Fig. 3). Thus, domains C and E are highly conserved among all reptilian ERs, whereas A/B, D and F domains show greater variability. Phylogenetic analyses of ERα sequences were generally consistent with existing phylogenetic hypothesis regarding vertebrate relationships (e.g., Carroll, 1988; Katsu et al., 2004). To better understand the position of crocodile ERα protein in the evolutionary history of the ERα protein and their reciprocal relationship, a phylogenetic tree was constructed using Phylip software using numerous ERα and ERβ proteins from various vertebrates (Fig. 4). The result shows that the alligator, caiman and crocodile ERα proteins were more similar to avian ERα than to other reptiles (Fig. 4). The similarity in predicted protein sequence for the DNA binding region of ERα was identical whereas that for the ligand-binding region was 99–100% similar, a surprising degree of similarity for a group that had a last common ancestor almost 83 mya. The amino acid sequence of crocodile ERα DNA-binding domain is 100% identical to alligator, chicken and human, however, the similarity of the nucleic acid corresponding to DNA-binding domain is 98% identical to alligator, 91% to chicken and 87% to human (data not shown). We should examine the molecular evolution with both nucleic acids and deduced amino acids.

ATGACCATGACCCTTCACACCAAAACCTCTGGAGTTACTCTGCTGCACCAGATTCAAGGCACTGAACTGGAGACTTTGAGCAGACCTCAGCTGAAGATTCCCTTAGATCGTTCGCTCACT M T M T L H T K T S G V T L L H Q I Q G T E L E T L S R P Q L K I P L D R S L T

120 40

GAGATGTATGTGGAGAGCAACAAGACAGGCATTTTTAACTACCCAGAAGGCACCACTTACGATTTTGCCACTGCTGCTCCAGTGTACAGCTCTACTAGCCTCAGTTATGCCCCTACTTCT E M Y V E S N K T G I F N Y P E G T T Y D F A T A A P V Y S S T S L S Y A P T S

240 80

GAATCATATGGATCCAGCAGTTTGGGAGGGTTTCATTCACTGAACAATGTCCCACCGAGCCCAGTGGTGTTCTTACAAACTGCGCCCCAGCTCTCTCCGTTCATTCATCACCATAGCCAA E S Y G S S S L G G F H S L N N V P P S P V V F L Q T A P Q L S P F I H H H S Q

360 120

CAAGTACCGTACTACCTTGAAAATGATCAAAGCGGCTTTGGAATGAGGGAAGCTGCCCCTCCAACTTTTTACAGGCCAGGTGCAGATAACAGGCGTCAGAGTGGCAGGGAGAGGATGTCC Q V P Y Y L E N D Q S G F G M R E A A P P T F Y R P G A D N R R Q S G R E R M S

480 160

AGCACCAGTGAAAAAGCGAGCCTGTCCATGGAATCCACAAAGGAGACCCGGTATTGTGCTGTGTGCAATGACTATGCTTCAGGCTACCATTATGGAGTTTGGTCTTGTGAGGGCTGTAAG S T S E K A S L S M E S T K E T R Y C A V C N D Y A S G Y H Y G V W S C E G C K

600 200

GCTTTCTTCAAAAGAAGTATTCAAGGGCACAATGACTACATGTGTCCTGCTACTAATCAGTGTACCATTGACAAGAACCGGAGAAAGAGCTGCCAAGCTTGCCGACTACGAAAGTGCTAT A F F K R S I Q G H N D Y M C P A T N Q C T I D K N R R K S C Q A C R L R K C Y

720 240

GAAGTGGGAATGATGAAAGGTGGGATCCGAAAAGACCGGAGAGGTGGGCGTATGTTGAAACAAAAACGCCAAAGAGAGGAGCAGGATGCCAGAAATGGAGAAACTGCTACTGCTGATATG E V G M M K G G I R K D R R G G R M L K Q K R Q R E E Q D A R N G E T A T A D M

840 280

AGAACCCCCACCCTCTGGACAAGTCCACTTGTGATTAAGCATACTAAGAAGAATAGTCCAGCCCTATCCCTGACAGCAGAGCAGATGGTCAGTGCCTTGCTGGAAGCTGAGCCTCCCATA R T P T L W T S P L V I K H T K K N S P A L S L T A E Q M V S A L L E A E P P I

960 320

GTCTATTCTGAGTATGACCCAAACAGACCATTCAATGAAGCCTCTATGATGACCCTGTTGACAAACCTTGCAGACCGAGAACTTGTGCACATGATCAACTGGGCAAAGAGAGTGCCAGGT 1080 V Y S E Y D P N R P F N E A S M M T L L T N L A D R E L V H M I N W A K R V P G 360 TTTGTGGATTTAACACTCCACGATCAGGTCCATCTACTGGAATGTGCCTGGTTAGAGATACTCATGATTGGCTTAGTCTGGCGTTCAATGGAACATCCAGGAAAGCTCTTATTTGCACCT 1200 F V D L T L H D Q V H L L E C A W L E I L M I G L V W R S M E H P G K L L F A P 400 AATCTATTACTAGACAGAAATCAAGGGAAGTGTGTGGAAGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACTGCTGCTCGATTTCGTATGATGAATCTCCAGGGGGAGGAATTTGTG 1320 N L L L D R N Q G K C V E G M V E I F D M L L A T A A R F R M M N L Q G E E F V 440 TGCCTTAAGTCTATCATTCTGCTCAATTCTGGTGTATATACCTTTCTTTCCAGCACCTTGAAATCTCTGGAAGAAAAGGACTATATTCATCGTGTTCTGGACAAAATTACAGATACTCTG 1440 C L K S I I L L N S G V Y T F L S S T L K S L E E K D Y I H R V L D K I T D T L 480 ATTCACTTAATGGCCAAGTCAGGTCTTTCTCTGCAGCAGCAACACAGGCGACTGGCTCAGCTTCTCCTCATCCTTTCACACATCAGGCACATGAGCAATAAAGGGATGGAGCACCTGTAC 1560 I H L M A K S G L S L Q Q Q H R R L A Q L L L I L S H I R H M S N K G M E H L Y 520 AACATGAAGTGCAAAAATGTAGTACCTCTTTATGATTTGTTGCTGGAGATGCTAGATGCTCACCGACTGCATGCCCCAGCAGCTAGAAATGCTGCCCAAGTAGAAGAGGAGACTCGGCTG 1680 N M K C K N V V P L Y D L L L E M L D A H R L H A P A A R N A A Q V E E E T R L 560 ACAACTGCATCAGCTTCATCGCATTCCTTGCAGTCATTTTACATAAACAACAGGGAAGATGAGAATTTGCAAAACACAATATGA 1764 T T A S A S S H S L Q S F Y I N N R E D E N L Q N T I * 587

Fig. 1. Nucleotide sequence of ERα cDNA and the deduced amino acid sequence for the Nile crocodile (Crocodylus niloticus). The amplified DNA fragment of the first PCR using the degenerate primer set present in the Materials and methods is indicated by the box. The numbers on the right refer to the position of the nucleotides and the amino acids.

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A/B domain alligator caiman crocodile chicken lizard Xenopus

MTMTLHTKTSGVTLLHQIQGTELETLSRPQLKIPLDRSLSEMYVESNKTGIFNYPEGTTYDFATAAP-VYSSTSLSYAPTSESYGSSSLGGFHSLNNVPPSPVVFLQTAPQLSPFIHHHS MTMTLHTKTSGVTLLHQIQGTELETLSRPQLKIPLDRSLSEMYVENNKTGIFNYPEGTTYDFATAAP-VYSSTSLSYAPTSESYGSSSLGGFHSLNNVPPSPVVFLQTAPQLSPFVHHHS MTMTLHTKTSGVTLLHQIQGTELETLSRPQLKIPLDRSLTEMYVESNKTGIFNYPEGTTYDFATAAP-VYSSTSLSYAPTSESYGSSSLGGFHSLNNVPPSPVVFLQTAPQLSPFIHHHS MTMTLHTKASGVTLLHQIQGTELETLSRPQLKIPLERSLSDMYVESNKTGVFNYPEGATYDFGTTAP-VYGSTTLSYAPTSESFGSSSLAGFHSLNNVPPSPVVFLQTAPQLSPFIHHHS MTMTLHTKTSGVALLHQIQGSELEPLNRPQLKIPLERPISEMYVDSNKTGVFNYPEGATYDFSTAAP-VYSSASLSYASTNESFGSGNLGGLHSLNNVPPSPVVFLQTAPQLSPFIHHHN MTMPLPNKTTGVTFLHQIQSSELETLTRPPLKISLERPLGEMYVENNRTGIFNYPEGTTYDFAAAAAPVYSSASLSYAASSETFGSSSLTGLHTLNNVPPSPVVFLQTP-QLSPFIHHHG *** * * ** ***** *** * ** *** * * *** * ** ****** **** * ** * **** * ** * * * ************** ***** ***

DNA-binding domain alligator caiman crocodile chicken lizard Xenopus

QQVPYYLENDQSGFGMREAAPSTFYRPGADSRRQSGRERMSSTSEKTSLSMESTKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC QQVPYYLENDQSGFGMREAASSTFYRPSADSRHQSGRERMSSTSEKASLSMESTKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC QQVPYYLENDQSGFGMREAAPPTFYRPGADNRRQSGRERMSSTSEKASLSMESTKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC QQVPYYLENEQGSFGMREAAPPAFYRPSSDNRRHSIRERMSSTNEKGSLSMESTKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC QQVPYYLENEPSSSAMREAFPTAFYRPGSENRHHGGR---ASNSEKGSLSMESTKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC QQVPYYLESEQGTFAVREAAPPTFYRSSSDNRRQSGRERMSSANDKGPPSMESTKETRYCAVCSDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC ******** *** *** * * * * ************** ********************************************************

D domain alligator caiman crocodile chicken lizard Xenopus

YEVGMMKGGIRKDRRGGRMLKQKRQREEQDARNGETATAEMRTPTLWTSPLVIKHTKKNSPALSLTAEQMVSALLEAEPPIVYSEYDPNRPFNEASMMTLLTNLADRELVHMINWAKRVP YEVGMMKGGIRKDRRGGRMLKQKRQREEQDARNGETATAEMRTPTLWTSPLVIKHTKKNSPALSLTAEQMVSALLEAEPPIVYSEYDPNRPFNEASMMTLLTNLADRELVHMINWAKRVP YEVGMMKGGIRKDRRGGRMLKQKRQREEQDARNGETATADMRTPTLWTSPLVIKHTKKNSPALSLTAEQMVSALLEAEPPIVYSEYDPNRPFNEASMMTLLTNLADRELVHMINWAKRVP YEVGMMKGGIRKDRRGGEMMKQKRQREEQDSRNGEASSTELRAPTLWTSPLVVKHNKKNSPALSLTAEQMVSALLEAEPPIVYSEYDPNRPFNEASMMTLLTNLADRELVHMINWAKRVP YEVGMMKGGIRKDRRGGRMLKHKRQRDELDGRN-AVAVTEARNTTLWPSPLMIKHSKKNSPALSLTAEQMVSALLDAEPPIVYSEYDPSSPFSEASVMTLLTNLADRELVHMITWAKRVP YEVGMMKGGIRKDRRGGRMLKHKRQKEEQEQKN-DVDPSEIRTASIWVN--PSVKSMKLSPVLSLTAEQLISALMEAEPPIVYSEHDSTKPLSEASMMTLLTNLADKELVHMINWAKRVP ***************** * * *** * * * * * ** ******* *** ********* * * *** ********* ****** ******

alligator caiman crocodile chicken lizard Xenopus

GFVDLTLHDQVHLLECAWLEILMIGLVWRSVEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATAARFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDYIHRVLDKITDT GFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATAARFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDYIHRVLDKITDT GFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATAARFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDYIHRVLDKITDT GFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATAARFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEERDYIHRVLDKITDT GFVDLALHDQVHLLECAWLEILMIGLIWRSLEHPGKLLFAPNLLLDRSQGMCVEGFVEIFDMLLATSSRFRMMNIQGEEFVCLKSIILLNSGIYTFLSSTLRSLEEKEHIHRVLDKITDT GFVDLTLHDQVHLLECAWLEILMVGLIWRSVEHPGKLSFAPNLLLDRNQGRCVEGLVEIFDMLVTTATRFRMMRLRGEEFICLKSIILLNSGVYTFLSSTLESLEDTDLIHIILDKIIDT ***** ***************** ** *** ****** ********* ** **** ******* * ***** **** *********** ******** *** ** **** **

alligator caiman crocodile chicken lizard Xenopus

LIHLMAKSGLSLQQQHRRLAQLLLILSHIRHMSNKGMEHLYNMKCKNVVPLYDLLLEMLDAHRLHAPAARNAAQVEEETR--LTTASASSHSLQSFYINNREDENLQNTI LIHLMAKSGLSLQQQHRRLAQLLLILSHIRHMSNKGMEHLYNMKCKNVVPLYDLLLEMLDAHRLHAPAARNAAQVEEETR--LTTASASSHSLQSFYINNREDENLQNTI LIHLMAKSGLSLQQQHRRLAQLLLILSHIRHMSNKGMEHLYNMKCKNVVPLYDLLLEMLDAHRLHAPAARNAAQVEEETR--LTTASASSHSLQSFYINNREDENLQNTI LIHLMAKSGLSLQQQHRRLAQLLLILSHIRHMSNKGMEHLYNMKCKNVVPLYDLLLEMLDAHRLHAPAARSAAPMEEENRNQLTTAPASSHSLQSFYINSKEEESMQNTI LTHLMAKSGLSLQQQHRRLAQLLLMLSHIRHMSNKGMEHLYNMKCKNVVPLYDLLLEMLDAHRLHERRTPTSEQAMNQLTN----ASTSVHSLPPCYVNKREEENEQEAV LVHFMAKSGLSLQQQQRRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRIHTPKDKTTTQEEDSRSPPTTTVNGASPCLQPYYTNTEEVSLQSTV* * *********** ******** **************** ********************* * * * * *

Ligand-binding domain

F domain

Fig. 2. Aligned deduced amino acid sequences of Nile crocodile ERα with that of the American alligator (AB115909), caiman (AB055220), chicken (X03805), lizard (AB055221) and Xenopus (L20735). Asterisks indicate identical amino acid residues. Gaps (−) were introduced to optimize the sequence alignment. The putative A/B to F domains are indicated above the sequences.

1

crocodile

C

97

95

lizard

77

chicken

Crocodile Alligator Caiman Lizard Chicken

100

87

Crocodile 100%

100

79

100

Alligator 98% 100%

91

88

45

99

Lizard 82% 82% 81% 100%

4. Discussion

581

589

539

345

Caiman 98% 98% 100%

587

100 535

341

245

179

1

587

100 539

345 99

242

176

1

99

245 100

F 539

99

179

1

caiman

E 345

245 100

587

539

345 D

179

1

alligator

245

179 A/B

Expression of ERα in the gonad of male and female Nile crocodiles was examined. RT-PCR revealed the expression of ERα mRNA in both male and female juvenile gonads. There was no amplification of DNA product in the negative in control (RT−) that lacked the enzyme required as a template (Fig. 5).

76

Chicken 92% 91% 91% 81% 100%

Fig. 3. Domain structure of Nile crocodile ERα, and homology with ERα of the American alligator, caiman, lizard and chicken. The functional A/B to F domains are schematically represented with the numbers of amino acid residues indicated. The figures within each box indicate the percent homology of the domain relative to crocodile ERα. Note the high sequence similarity with the alligator, caiman, chicken and lizard.

Previously, three full sequences for ERα have been reported for reptiles, C. crocodilus and C. uniparens (Sumida et al., 2001), and A. mississippiensis (Katsu et al., 2004) as well as partial sequences from two freshwater turtles, T. scripta: (Bergeron et al., 1998) and C. picta: (Custodia-Lora and Callard, 2002a), the green anole, A. carolinensis: (Matthews and Zacharewski, 2000) and the whiptail lizard, C. uniparens: (Young et al., 1994). The full sequence for ERα reported here for the Nile crocodile is similar in sequence to that reported for A. mississippiensis (Katsu et al., 2004) and C. crocodylus (Sumida et al., 2001). Sumida et al. (2001) and Katsu et al. (2004) reported that the ERα from the caiman and the American alligator had an open reading frame 1764 base pairs in length,

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100

100

100

ERα ERβ 100

100

54

0.1 EAASS Support: 95-100% 90-95% 70-90% < 70%

100

Ictalurus punctatus (Channel Catfish) Oryzias latipes (Japanese Medaka) Salmo salar (Atlantic Salmon) 51 Micropogonias undulatus (Atlantic Croaker) 100 98 Sparus aurata (Gilthead Seabream) 100 30 Pagrus major (Red Seabream) Oreochromis niloticus (Nile Tilapia) 100 Oreochromis aureus (Blue Tilapia) Xenopus laevis (African Clawed Frog) 100 Xenopus tropicalis (Silurana tropicalis) Crocodylus niloticus (Nile crocodile) 57 100 Caiman crocodilus (Spectacled Caiman) Alligator mississippiensis (American Alligator) 53 Trachemys scripta (Red-eared Slider Turtle) 100 Taeniopygia guttata (Zebra Finch) 55 Gallus gallus (Chicken) 100 Coturnix Coturnix japonica (Japanese Quail:AAN63674) 98 Cnemidophorus uniparens (Whiptail:BAB79437) 94 Anolis carolinensis (Green Anole:Q9YHT3) 74 Homo sapiens (Human:P03372) 99 Sus scrofa (Pig:Q29040) 41 Mus musculus (House Mouse:P19785) 100 Rattus norvegicus (Noway Rat:P06211) Squalus acanthias (Spiny Dogfish) Carassius auratus (Goldfish) 83 Oreochromis niloticus (Nile Tilapia) 83 Sparus aurata (Gilthead Seabream) 96 Micropogonias undulatus (Atlantic Croaker) Micropogonias undulatus (Atlantic Croaker) 97 Anguilla japonica (Japanese Eel) Gallus gallus (Chicken) 80 Coturnix japonica (Japanese Quail) 81 93 Sturnus vulgaris (Common Starling) Alligator mississippiensis (American Alligator) Rattus norvegicus (Rat) 85 Mus musculus (House Mouse) 41 Homo sapiens (Human) 87 Bos taurus (Cow) 78 Sus scrofa (Pig)

Teleostean

Anuran

Reptilian & Avian

Mammalian Selachian Teleostean

Reptilian & Avian

Mammalian

Fig. 4. Phylogenic tree of vertebrate ERα and ERβ using deduced amino acid sequences of vertebrate ERα and ERβ˜ D-domain and ligand-binding domain of their sequence were used. The phylogenic tree was constructed using the Phylip computer program with the JTT matrix, neighbor-joining method and bootstrap re-sampling for 1000 times. The number indicates the bootstrap value (%), and the width of branch reflects the support calculated by the bootstrap re-sampling. The length of branch reflects estimated numbers of substitutions along each branch. The scale bar indicates 0.1 expected amino acid substitutions per site.

encoding a 587 amino acid protein, as we have reported here for the Nile crocodile (C. niloticus). A phylogenetic analysis reveals high similarity among the sequences of the three crocodilians and a close relationship with birds. The American alligator and Nile crocodile have been recognized as distinct species since the Pleistocene, or approximately 1.8 million years from the present (Brochu, 2003). The Nile crocodile belongs to a clade that includes the extant New World crocodiles. Alligators and caimen share phylogenetic history and thus it is not surprising to see a high identity among the ERα sequences of these species, but members of the Crocodyloidea (includes the modern crocodiles) and Alligatoroidea (includes the modern alligators and F

M

F

M

770 612 495 392

RT (+)

RT (-)

Fig. 5. Expression of ERα mRNA in gonads of juvenile Nile crocodiles. Total RNA was isolated from the gonads of juvenile crocodiles and reverse transcription-PCR was used to determine the expression of ERα. The cDNA was used as a template in RT (+), whereas the RT product reacted without enzyme, was used as the template for the RT (−) reaction.

caimen) branches of the Crocodylia have not shared an ancestor since the pre-Campanian period of the Cretaceous, or more than 83 mya (Brochu, 2003). Likewise, the alligators and caimen belong to the Alligatoroidea but shared a common ancestor sometime in the Paleocene or about 60 mya (Brochu, 2003). Our observations on the conserved sequence homology of ERα is not unique, as we recently observed very high sequence identity (97–99%) for two proto-oncogenes, c-Jun and DJ-1, obtained from the American alligator and Nile crocodile (Katsu et al., unpublished data). These data support our earlier proposal (see Katsu et al., 2004) that crocodilians exhibit slow evolution of its nuclear genome, with highly conserved sequence homology among species. The slower evolution for genes encoding steroid receptors and protooncogenes in crocodilians provides an interesting observation but its implications are not resolved, in part due to the relatively sparse data for other gene families and other reptiles. The conservation of sequence could imply that functionally relevant ancestral features have been retained but it might also indicate slower overall genomic evolution in the crocodilians. The apparent absence of “warm-blooded” isochore structure in reptilian lineages would provide support for this hypothesis (reviewed by Bernardi, 2000). Interestingly, mitochondrial genomes of crocodilians are very divergent suggesting that the slower rate of genomic change hypothesized here is clearly limited to the nuclear genome of these organisms.

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We observed that testis and ovary exhibit gene transcripts for ERα. This result is consistent with our analyses of alligator ERα expression in that we have detected this receptor in the juvenile gonad of both sexes (Katsu et al., 2004; Kohno et al., unpubl. data). Katsu et al. (2004) also observed that a single pharmacological injection of 17β-estradiol reduced ERα mRNA expression, but that ERβ mRNA expression was unaffected in the ovary of juvenile alligators within 30 h of treatment. Exogenous 17β-estradiol did not depress ERβ or progesterone receptor mRNA levels in the same animals. We have tried to isolate the cDNA clone of Nile crocodile ERβ, however, we have been unsuccessful. Future studies need to examine the regulation of ERαand ERβ in detail and focus on ontogenic and sexually dimorphic responses in crocodilians, including the Nile crocodile. In addition to examining the interaction of ERα with endogenous steroids, work must also examine their possible role of environmental contaminants affecting the developmental and reproductive biology of Nile crocodiles. Various environmental contaminants, in particular DDT and its metabolites, bind the alligator ER (Guillette et al., 2002; Vonier et al., 1996). Given the high sequence homology between the alligator and crocodile ligand-binding regions of ERα, it is almost assured that the same compounds will act in a fashion in the Nile crocodile similar to that observed in the American alligator. For example, sex determination in developing turtle and alligator embryos can be altered (male to female) in species with TSD if they are exposed during a critical window of development to various pesticides and pesticide metabolites having estrogenic activity (Matter et al., 1998; Willingham and Crews, 1999; Milnes et al., 2005). Given the continued use of DDT in tropical regions of Africa with malaria threats, it is worth further study to examine possible interactions between this pesticide and its metabolites and crocodilian biology. These studies should examine the molecular interactions between the steroid receptors from this species and native ligands or contaminants. Acknowledgements We thank Mr. Albert Pretorius, Thaba Kwena Crocodile Farm, Republic of South Africa for assess to his farm, assistance by his workers during sample collection and donation of crocodile tissues used in this project. This work was supported in part by grants to LJG (UF Opportunity Fund), and YK and TI (Grant-in-Aid for Scientific Research from Ministry of Education, Science, Sports and Culture of Japan; grants from Ministry of Environment, Japan). References Bergeron, J.M., Gahr, M., Horan, K., Wibbels, T., Crews, D., 1998. Cloning and in situ hybridization analysis of estrogen receptor in the developing gonad of the red-eared slider turtle, a species with temperature-dependent sex determination. Dev. Growth Diff. 40, 243–254. Bernardi, G., 2000. Isochores and the evolutionary genomics of vertebrates. Gene 241, 3–17. Blumberg, B., Evans, R.M., 1998. Orphan nuclear receptors — new ligands and new possibilities. Genes Dev. 12, 3149–3155.

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