General and Comparative Endocrinology 138 (2004) 70–77

www.elsevier.com/locate/ygcen

Communications in Genomics and Proteomics

Molecular characterization of the leopard gecko POMC geneand expressional change in the testis by acclimation to low

temperature and with a short photoperiod

Daisuke Endo¤ and Min Kyun Park

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

Received 4 February 2004; revised 23 April 2004; accepted 26 April 2004Available online 4 June 2004

Abstract

The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and �-endorphin, was isolatedand analyzed in the squamata species, the leopard gecko, Eublepharis macularius. Leopard gecko POMC (lgPOMC) cDNA is com-posed of 1299 bp, excluding the poly(A) tail, and encodes 270 amino acids. The deduced amino acid sequence showed the same struc-tural organization as that of other species and displayed identity with those of other vertebrates: 68% with mud turtles, 57/57% withAfrican clawed frog A/B, 53% with chickens, and 45% with mice. In a phylogenic tree, the lgPOMC clustered with the sequences ofthe mud turtle POMC and python POMC. The lgPOMC gene comprises three exons and two introns and this structure is consistentwith humans, rats, mice, African clawed frog and zebraWsh. RT-PCR analysis revealed that the lgPOMC mRNA was expressed onlyin the whole brain, pituitary, and gonads. To analyze in more detail, a competitive assay system to quantify the expression levels ofPOMC mRNA was established. We measured the POMC mRNA expression levels in the leopard gecko testes following transferfrom a condition of 29 °C, 16L/8D to 18 °C, 10L/14D over 6 weeks. This 6-week acclimation increased the POMC mRNA expressionlevels signiWcantly. This suggests that the leopard gecko POMC-derived peptides play a role in the mediation of the eVect of environ-mental factors on reproduction. 2004 Elsevier Inc. All rights reserved.

1. Introduction

Proopiomelanocortin (POMC) is the common pre-cursor of corticotropin, �-endorphin, and melanotro-pins. These peptide hormones play an important role instress response, skin pigmentation, thermoregulation,and reproduction (Hadley and Haskell-Luevano, 1999).The POMC gene is mainly expressed in the anterior andintermediate lobes of the pituitary gland and in thehypothalamus. The functional peptides are generatedthrough a series of ordered proteolytic cleavages atpaired basic residues of the POMC by pro-hormone con-vertase, such as PC1 and PC2, displaying a cell-type-spe-ciWc pattern (Benjannet et al., 1991).

The POMC gene and its derived peptides are alsoexpressed in many peripheral tissues, such as the kidney

¤ Corresponding author. Fax: +81-3-5841-4439.E-mail address: MS7D-ENDU@asahi-net.or.jp (D. Endo).

0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2004.04.009

(Takeuchi et al., 1999), liver (DeBold et al., 1988; Varsa-mos et al., 2003), adrenal gland (DeBold et al., 1988;Takeuchi et al., 1999), and skin (DeBold et al., 1988;Farooqui et al., 1993; Shen et al., 2003). The pattern ofexpression diVered between species. For example,POMC mRNA is expressed in the liver of mice and seabass (DeBold et al., 1988; Varsamos et al., 2003), but notin the liver of chickens and mud turtles (Shen et al., 2003;Takeuchi et al., 1999). In the testis and ovary, however,POMC gene expression is reported in species from allvertebrate classes (DeBold et al., 1988; Nabissi et al.,1998; Takeuchi et al., 1999; Varsamos et al., 2003). Thissuggested that POMC-derived peptides have a highlyconserved function in regulating reproduction. Recently,many studies have been conducted regarding the role ofPOMC-derived peptides in the gonads via autocrineand/or paracrine mechanisms. In the ovary of the lizard,Podarcis s. sicula Raf, �-endorphin stimulates follicularestrogen production in vitro (Polzonetti-Magni et al.,

D. Endo, M.K. Park / General and Comparative Endocrinology 138 (2004) 70–77 71

1994). Naltrexone, the antagonist of the opioid receptor,increased the androgen level in the testis of the frog,Rana esculenta (Facchinetti et al., 1993). Not only �-endorphin but also the adrenocorticotropic hormone(ACTH) was reported to stimulate testosterone produc-tion directly in the testis of some mammalian species, forexample, rabbits and guinea pigs (Juniewicz et al., 1988).There are also a few reports that the mRNA expressionlevels of POMC were aVected by stress and the photope-riod in Rana esculenta (Nabissi et al., 2001) and Syrianhamsters (Morgan et al., 2003), respectively. This sug-gests that POMC is an important molecule mediatingthe eVect of environmental factors on reproductive phe-nomena.

Squamata are found throughout the world, andacquire various characteristics to adapt to their diverseenvironments. For successful adaptation, the reproduc-tive endocrine system will attain the most eYcient repro-ductive activity. Squamata are often used to study theWeld of environmental endocrinology. In previousresearch, annual changes in the weight of gonads and theconcentration of circulating sex steroid hormones, havebeen reported in some species (Arslan et al., 1978;Bourne and Seamark, 1975; Courty and Dufaure, 1979;Licht, 1984) and the eVects of ambient environmentalfactors, such as temperature and light, have also beenextensively studied (Borrelli et al., 2000; Gavaud, 1991;Licht, 1969, 1971; Marion, 1970; Sexton et al., 1971).However, the molecular mechanisms underlying suchcomplex traits are still not generally understood in squa-mata animals.

The leopard gecko, Eublepharis macularius, is easilytamed, and is easy to breed in the laboratory, unlikelyother squamata. In this animal, the incubation tempera-ture during embryonic development determines theirgonadal sex (Viets et al., 1993). The leopard gecko hasalso been described as a seasonal breeder (Vosjoli et al.,1998), and investigations into the eVect of environmentalfactors on the expression of sex steroid hormone recep-tors have been reported. The expression levels of theandrogen receptor mRNA was up-regulated by a 6-weekacclimation to low temperature and a short photoperiod(Endo and Park, 2003). These results suggest that theleopard gecko is expected to be a useful experimentalmodel.

The characterization of the leopard gecko POMCgene will be a useful Wrst step to investigate the molecu-lar mechanism that mediates the eVect of environmentalfactors on reproduction. There is only one report on thesequence of full-length cDNA of turtle POMC in reptilesand no reports on squamata species. Such informationwould therefore add to our understanding of POMCphylogeny. We therefore cloned leopard gecko POMCcDNA and characterized this gene and the eVect of envi-ronmental factors on the expression of the POMCmRNA was examined.

2. Materials and methods

2.1. Animals

Fertile male and female leopard geckos of 15 monthsof age were used. They were hatched at 29 °C and raisedat same temperature. Crickets were provided as the maindiet three times per week and shelter, water, and a cal-cium supplement were supplied ad libitum. Animalswere maintained on a 14:10 h light/dark cycle at 29 °C(Group cont, n D 3). The short photoperiod and low tem-perature acclimation was then implemented on an 8:16 hlight/dark cycle at 18 °C for 6 weeks (Group A, n D 3).For some animals, these environmental conditions werereturned to the initial conditions (14:10 h light/darkcycle, 29 °C), which were maintained for 2 weeks (GroupB, n D 3). All environmental changes were carried outstepwise over a week to minimize stress. An animal wasanesthetized with sodium pentobarbital (50 mg/kg) andkilled by rapid decapitation, followed by complete bleed-ing. The tissues, the testis from male adult leopardgeckos, and the ovaries, whole brain, pituitary gland,spleen, adrenal gland, kidney, liver, and skin from thefemale adult leopard gecko were quickly removed andfrozen immediately in liquid nitrogen and stored at¡70 °C until used.

2.2. RNA extraction and cDNA synthesis

Total RNA was extracted using ISOGEN (NipponGene, Tokyo, Japan). Male whole brain cDNA used forrapid ampliWcation of cDNA ends (RACE; see Section2.3) was synthesized from 3 �g of total RNA using aSMART RACE cDNA AmpliWcation Kit (BD Bio-sciences Clontech, Palo Alto, CA), according to the man-ufacturer’s instructions. The cDNAs used as templatesfor RT-PCR were synthesized from 3 �g of denaturedtotal RNA using 5 �M oligo(dT) primer and 100 U ofM-MLV Reverse transcriptase (Promega, Madison, WI)in a 20-�l reaction volume with incubation at 42 °C for1.5 h.

2.3. Molecular cloning of POMC cDNA by RACE andRT-PCR

RT-PCR was carried out to obtain a partial POMCcDNA from male whole brain cDNA using degenerateprimers, dege-SE01 and dege-AS01 (deduced from aconserved region of �-MSH and �-endorphin, respec-tively). All of the following PCR ampliWcations wereperformed in a 20-�l reaction mixture containing eachprimer at 1�M, 0.25 U of TaKaRa Ex Taq (TaKaRa,Shiga, Japan), each dNTP at 250 �M and Ex Taq BuVer(TaKaRa). The reaction conditions for RT-PCR were asfollows: 94 °C for 5 min, 35 cycles of 94 °C for 40 s, 58 °Cfor 30 s, 72 °C for 1 min, and 72 °C for 7 min. The amp-

72 D. Endo, M.K. Park / General and Comparative Endocrinology 138 (2004) 70–77

liWed products were separated by electrophoresis in a1.5% agarose gel and visualized using ethidium bromidestaining. DNA fragments were extracted using a QIAquick Gel Extraction Kit (Qiagen, K.K., Tokyo, Japan)and directly sequenced using a dRhodamine TerminatorCycle Sequencing FS Ready Reaction Kit (Applied Bio-systems, Tokyo, Japan).

After determining the partial sequence of POMCcDNA, sense, and antisense gene-speciWc primers weredesigned based on the sequence. RACE was carried out toobtain the complete sequence. 3�- and 5�-RACE was per-formed with SE01 and Nested Universal Primer (NUP),and NUP and AS01, respectively. Each PCR conditionwas as follows: 94°C for 5min, 35 cycles of 94 °C for 40 s,64°C for 30 s, 72 °C for 1min, and 72 °C for 7min. TheampliWed products were sequenced as described above.

The sequence of the entire open reading frame wasconWrmed with the gene-speciWc primers, 5�-UTR-SE01and 3�-UTR-AS01 using the cDNAs of whole brainsfrom three individuals.

2.4. Genomic structure of the POMC gene

Genomic DNA was extracted from the liver usingISOGEN. The POMC gene was cloned using severalprimer pairs. Each PCR condition was as follows: 94 °Cfor 5 min, 35 cycles of 94 °C for 40 s, 64 °C for 30 s, 72 °Cfor 1 min, and 72 °C for 7 min. The ampliWed productswere sequenced as described above.

2.5. Comparison of the amino acid sequences of variousPOMCs

The CLUSTAL X program (version 1.81) (Thomp-son et al., 1997) was used with default settings to alignthe deduced amino acid sequences of the POMC of theleopard gecko and other species with each other. Theamino acid identity was calculated between entire ORFs,signal peptides, �-MSH, �-MSH, ACTH, �-MSH, and�-endorphin, respectively, using GeneDoc software(version 2.6.002) (Nicholas and Nicholas, 1997).

2.6. Molecular phylogenic analysis

The amino acid sequences of the entire ORFs of thePOMC in the leopard gecko and several species from allvertebrate classes were aligned using CLUSTAL X withdefault settings. The alignment of the amino acidsequences was used to generate the phylogenic tree. Theneighbor-joining method (Saitou and Nei, 1987) wasused. The proopiomelanotropin (POM) and proopiocor-tin (POC) of the sea lamprey were used as outgroups(Takahashi et al., 2001). These genes are thought toevolve from same ancestral gene as POMC. Bootstrapvalues were calculated with 1000 replications to estimatethe robustness of internal branches. The GenBank acces-

sion numbers of the POMC used in the comparisons andphylogenic analysis are as follows: human P01189;mouse P01193; gray short-tailed opossum AY056476;chicken AB019555; python AF369042; mud turtleAY099290; xenopus-A X03843; xenopus-B X03844;bullfrog X15510; African lungWsh AF100164; Japaneseeel AY158010; tilapia AF116240; zebraWsh AY125332;white sturgeon-A AF092937; white sturgeon-BAF092936; polypterus senegals AF465781; port jacksonshark AY195737; sea lamprey POM D5562 and sea lam-prey POC D55628.

2.7. Expression analysis

RT-PCR was performed to identify the possiblesource of the POMC-derived peptides. Twenty Wve nan-ograms of the cDNAs from the whole brain, ovary, tes-tis, adrenal gland, liver, heart, lung, and skin, 5 ng fromthe pituitary and genomic DNA were ampliWed usingspeciWc primers for the POMC, and �-actin (Table 1).The PCR conditions were as follows: 94 °C for 5 min, 35cycles of 94 °C for 40 s, 64 °C for 30 s, 72 °C for 1 min,and 72 °C for 7 min. The ampliWed products were elec-trophoresed on 1.5% agarose gel and stained with ethi-dium bromide. The primers for the POMC mRNA weredesigned to span intron 2 (Fig. 1) and no band wasdetected using the genomic DNA of the leopard geckounder PCR conditions (Fig. 4). The speciWcity of thePCR was conWrmed by sequence analysis.

2.8. EVect of the low temperature and a short photoperiod

A competitive PCR system was established accordingto the report of Endo and Park (2003) for quantifyingthe mRNA expression levels of POMC. Nine leopardgeckos were divided into three groups (Group Cont, A,and B) and treated as described above. The mRNAexpression levels of POMC were quantiWed in the testesof geckos from three groups. One arbitrary unit (a.u.)was deWned as an average of the mRNA expressionlevels in the testes of Group Cont.

2.9. Statistical analysis

Analysis of variance (ANOVA) followed by Welch’stest was used to analyze the data, where P 0 0.05 wasconsidered statistically signiWcant. The numbers of sam-ples are shown in parentheses (Table 2).

3. Results

3.1. Cloning leopard gecko POMC cDNA

We designed a degenerate primer based on thesequence of highly conserved regions in �-MSH and

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Table 2The eVect of low temperature and a short photoperiod on the mRNAexpression levels of POMC

Values are means (§) SE: group with asterisk is signiWcantly(P 0 0.05) diVerent from other groups. The numbers of samples areshown in parentheses.

Cont (3) Group A (3) Group B (3)

Relative expression level (a.u.)

1.00 § 0.79 9.55 § 3.06* 2.00 § 0.833

�-endorphin (Table 1). Using these primers, 267 bpof the partial sequence were acquired. Based on thispartial sequence, primers for 3�- and 5�-RACE, SE01and AS01, respectively, were designed (Table 1). 3�- and5�-RACE PCR resulted in one distinct band each. Thebands were electrophoresed, extracted, and sequenceddirectly using the primers SE01, SE02, AS01, AS02, andAS03. The length of the leopard gecko POMC cDNAand deduced amino acid was 1299 bp except the poly(A)tail with an open reading frame encoding a 270 amino

Table 1Oligonucleotid primers used for RACE, RT-PCR, genomic structure determination, and competitive-PCR

a Abbreviations for degenerate nucleotides: Y, C or T; R, G or A; K, G or T. N represents all four nucleotides.

Name Nucleotide sequencea Usage and location

dege-SE01 5�-ATGGARCAYTTYCGNTGGGG-3� For degenerate PCRdege-AS01 5�-CATRAANCCNCCRTANCKYTT-3� For degenerate PCRSE01 5�-CCTGTGAGGGTCTACCCAAATGGAG-3� For 3�-RACEAS01 5�-GTTCCACCGGAAGTGACGCATCTTG-3� For 5�-RACESE02 5�-GAATGCCATCATCAAAACCGCCTAC-3� For sequenceAS02 5�-GTCTTTCCAGGTCTTGTCTTTCCAGG-3� For sequence, RT-PCR and competitive-PCRSE03 5�-CTTAGGGAACACTTCATTCTGGTTC-3� For sequenceAS03 5�-TGACTCGGCTGAGAGATCCAGTCTG-3� For sequence and intron 2 (in exon 3)5�-UTR-SE01 5�-TCTACAGACCACACATTACGGCAAC-3� For conWrmation of sequence and for intron 1(in exon 1)3�-UTR-AS01 5�-ATTCAGAACCAGAATGAAGTGTTCCC-3� For conWrmation of sequenceSE04 5�-TTGCTGCACTTCCTCCTAAAGATGC-3� For intron 2, RT-PCR and competitive-PCR (exon 2)AS04 5�-CTTCTGTGTCCATATCCCTACAGCG-3� For intron 1 (exon 2)NUP 5�-AAGCAGTGGTATCAACGCAGAGT-3� For 3�- and 5�-RACECOMP-AS01 5�-GTCTTTCCAGGTCTTGTCTTTCCAGGCCTAAGTC

ACCACCGCTGCTGTTGC-3�

For construction of the competitor

Fig. 1. Schematic representation of the gene, cDNA, and predicted protein structure of leopard gecko POMC. (A) Structural organization of theleopard gecko POMC gene. The coding regions for POMC are indicated by boxes. The solid arrowheads show the positions of the primers for RT-PCR used in this study. (B) Structure of leopard gecko POMC cDNA. The open reading frame is indicated by a box. (C) Structure of the predictedleopard gecko POMC. The positions of the peptides probably generated by processing the POMC are indicated. The open and solid arrowheads indi-cate the dibasic and monobasic cleavage sites, respectively.

74 D. Endo, M.K. Park / General and Comparative Endocrinology 138 (2004) 70–77

acid prepropeptide. A polyadenylation signal was pres-ent in the 3�-untranslated region (UTR). The sequence ofthe full-length cDNA clone was deposited in GenBank(Accession No. AB128826).

3.2. Genomic structure

Using several primers, the genomic DNA of theleopard gecko was ampliWed. Using the primer set 5�-UTR-SE01 and AS04, a band of a diVerent size fromthe band ampliWed using the cDNA as template wasacquired. Intron 1 was identiWed by sequencing thisband. Intron 2 was also identiWed by the same methodusing the primers SE04 and AS03. The leopard geckoPOMC gene is comprised of 3 exons and 2 introns(Fig. 1). Exon 1 contains the 5�-UTR and has a lengthof 76 bp. Exon 2 contains the rest of 5�-UTR, encodesthe signal peptide and has a length of 156 bp. Exon 3,1063 bp, encodes 3 melanotropins and 1 corticotropinand 1 opioid. Introns 1 and 2 have lengths of about 2.7and 3.4 kb, respectively. The structure of the leopardgecko POMC gene is consistent with that of allreported species, such as humans (Takahashi et al.,1983), mice (Uhler et al., 1983), rats (Drouin et al.,1985), African clawed frog (Deen et al., 1992), andzebraWsh (Hansen et al., 2003).

3.3. Structure of the POMC and comparison with othervertebrate POMCs

The deduced amino acid sequence displayed identitywith other vertebrate: 68% with mud turtles, 57/57% withAfrican clawed frog (A/B), 53% with chickens, and 45%with mice. Fig. 2 shows a comparison of the amino acidsequence of the leopard gecko POMC precursor with thePOMC of the python, mud turtle, chicken, mouse, Africanclawed frog A and zebraWsh. Eight cleavage sites werefound in the leopard gecko POMC. C-terminal dibasicresidues for �-MSH are altered to Lys-Ser. However, thissite contains the rules that govern monobasic cleavages(Devi, 1991). All amino acid sequences of MSHs have theinvariant amino acid sequence, His-Phe-Arg-Tyr and thatof �-endorphin has a common N-terminal sequence (Tyr-Gly-Gly-Phe-Met) of a family of endogenous neuromod-ulators. No insertion was observed in the sequence of the�-MSH of the python (Venkatesh et al., 2001). A substitu-tion in �-MSH reported in Anolis carolinansis was alsoobserved (Dores et al., 1991).

3.4. Molecular phylogenic analysis

The phylogenic tree was calculated using an aminoacid alignment of the POMC sequences of 18 selected

Fig. 2. Alignment of the predicted amino acid sequence of leopard gecko POMC with diVerent species. The leopard gecko POMC sequence was com-pared to the python (only partial sequence available), mud turtle, chicken, mouse, Xenopus (A), and zebraWsh POMC. The identical residues with theleopard gecko POMC are shaded. Missing residues are shown by bars. The dibasic and monobasic cleavage sites are heavily shaded. The componentpeptides are indicated by over lines.

D. Endo, M.K. Park / General and Comparative Endocrinology 138 (2004) 70–77 75

species representing every class of vertebrate. The resultis shown in Fig. 3. As expected, the leopard geckoPOMC clustered with the sequences of reptiles, but thechicken POMC did not cluster with the reptiles.

3.5. Expression analysis

The tissue distribution of POMC mRNA was investi-gated by RT-PCR using the primers SE04 and AS02. Theprimer set is designed to span the exon/intron boundary,and the eVect of genomic DNA contamination could beexcluded. The leopard gecko POMC mRNA wasexpressed in the pituitary, whole brain, ovary, and testisbut not in the other organs examined (Fig. 4).

3.6. EVect of low temperature and a short photoperiod

In the testes, the expression levels of the POMC sig-niWcantly increased when the conditions were 18 °C, 8L/

Fig. 4. Expression analysis of POMC mRNA in various tissues of theleopard gecko. Five nanograms of cDNA pituitary, 25 ng of cDNAfrom the whole brain, pituitary, heart, kidney, liver, spleen, adrenalgland, skin, ovary and testis, total RNA without reverse transcription,and genomic DNA were subjected to PCR for leopard gecko POMCand �-actin. The testes were removed from the male adult leopardgecko and other tissues were removed from the female adult leopardgecko.

16D (P 00.05). This increased expression level decreasedto near the levels of the control 2 weeks after the condi-tions were returned to 29 °C, 14L/10D (Table 2).

4. Discussion

To investigate the mechanism of adaptation to theenvironment of reptiles is important because of theirunique position in vertebrate evolution. It is the onlyanimal class belonging to both amniotes and ectotherms.The diVerent mechanism of adaptation between endo-thermic amniotes (mammals and birds) and ectothermicamniotes (reptiles) is extremely interesting. We selectedleopard gecko, a lizard with temperature-dependent sexdetermination, as an experimental animal to initiateinvestigations into the molecular mechanisms of envi-ronmental control over reproduction. POMC is a pre-cursor of the hormones playing important roles in notonly the stress response but also reproduction. TheeVects of captive stress and a short photoperiod on theexpression of the POMC gene in the gonads have beenreported (Morgan et al., 2003; Nabissi et al., 2001). Theseresults suggested that the POMC is an important mole-cule in the environmental eVect on reproduction and wehad characterized the POMC gene in leopard gecko.

In the amino acid sequence of the leopard geckoPOMC cloned in this study, the basic structures of threemelanotropins, one endorphin, and one corticotropinwere conserved. The similarities among the overallamino acid sequences of the leopard gecko POMC andthose of other species were not particularly high, how-ever, in the sequences of the functional peptides, strongconservation was observed (Fig. 2). In the phylogenic

Fig. 3. Phylogenic tree of vertebrate POMC based on amino acid homology. The numbers at the branches represent the conWdence limits computedby the bootstrap procedure (1000 trials). For details on accession numbers in the GenBank databases, see Section 2.6.

76 D. Endo, M.K. Park / General and Comparative Endocrinology 138 (2004) 70–77

tree, the amino acid sequences of the POMC from reptil-ian species, including the leopard gecko, were clustered,although the sequences of chickens did not cluster withreptiles but mammals (Fig. 3). This result was not consis-tent with the phylogenic tree shown by another molecu-lar marker (Cao et al., 2000; Zardoya and Meyer, 2001).However, there were only two full lengths of cDNAsequences of reptilian species from two of Wve orders andone of avian species (Shen et al., 2003; Takeuchi et al.,1999). More sequence information is needed for furtherdiscussion.

The mRNA expressions of the leopard gecko POMCwere detected only in the whole brain, pituitary, andgonads, not in the tissues where the expressions werereported in other species. There is a report of expressionin the pancreas of another species of lizard (Putti et al.,1999), but no signal was detected in that of the leopardgecko. So, it is still unclear whether the functions in tis-sues other than the brain, pituitary and gonads are spe-cies speciWc or whether the expression is detected whenthe leopard gecko is exposed to stressors, such as thechange of environmental factors.

The expression levels of leopard gecko POMC in tes-tes were up-regulated by low temperature and a shortphotoperiod. This result suggests that the POMC-derived peptides play a role in the seasonality of testicu-lar activity. Many studies have been conducted about thefunctions of �-endorphin, POMC-derived peptides in thetesticular activity. For example, in the testis, �-endor-phin immunoreactivity showed seasonal variation, beingmost pronounced in the interstitial cells of sexually qui-escent lizards, Podarcis s. sicula Raf (Ciarcia et al., 1994).It has also been reported that �-endorphin decreasedboth basal and hCG stimulated testosterone release inthe testis of the rat directly in vitro (Kant and Saxena,1995). However, Naltrexone, the antagonist of the opi-oid receptor, increased the androgen level in the testis ofthe frog, R. esculenta (Facchinetti et al., 1993). And, itwas also reported that the infusion of synthetic humanACTH (1–24), another POMC-derived peptide, at a con-centration of 500 ng/ml increased testosterone secretionover that in non-treated controls in in vitro perfusedrabbit and guinea pig testes (Juniewicz et al., 1988). Inour previous study, no signiWcant change in the weightsof the testes and microscopic observation of the testicu-lar area was found by the same acclimation to low tem-perature and short photoperiod as this study (Endo andPark, 2003). Thus, the interpretation of this up-regula-tion of mRNA expression levels needs further study.

In this report, we cloned and characterized the leop-ard gecko POMC gene. This is the Wrst report on thecloning of a full-length sequence of POMC cDNA insquamata species and the genomic structure of thePOMC gene in reptiles. We found that the mRNAexpression levels of this gene in the testes were aVectedby changes in environmental factors. These results will

facilitate the understanding of the molecular mecha-nisms of the environmental control over reproduction inreptiles.

Acknowledgments

We are grateful to Prof. Y. Oka, Dr. M. Matsuda, Dr.Y. Akazome, Ms. M. Kyokuwa, Mr. M. Enomoto, andMr. T. Ikemoto, Department of Biological Sciences,Graduate School of Science, The University of Tokyo,for valuable discussions throughout this study.

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