最新性别决定研究论文

m th K e U d 4 ne 2 Sex steroid hormones are known to play a central role in vertebrate sex determination and differentiation. However, the tissues in which they are produced or received during development, especially around the period of sex determination of the gonads, have rarely among vertebrates. The existence of Sry on the male-spe- ous tubules of the testis with Sertoli cells and the expression induce incomplete sex reversal in marsupials and birds. on sex determination in reptilian species has also been reported (Wibbels and Crews, 1995; Ganesh et al., 1999). Demonstration of female-specific expression of P450arom in developing gonads also suggests the importance of sex * Corresponding author. Fax: +81 3 5841 4439. E-mail address: biopark@biol.s.u-tokyo.ac.jp (M.K. Park). Available online at www.sciencedirect.com General and Comparative Endocrin cific sex chromosome creates the gonads of embryo testes in mammals (Sinclair et al., 1990; Koopman et al., 1991), while Dmy is the sex-determining gene in medaka, a bony fish (Matsuda et al., 2002). Conversely, all crocodiles, most turtles, and some lizards have no sex chromosomes and sex is determined by incubation temperature rather than by a gene (Sarre et al., 2004). Sex steroid hormones, however, are known to play a pivotal role in sex determination and differentiation in all vertebrate classes downstream of the first determining switch. The ovaries of adult estrogen receptor a and b double knockout female mice exhibit fol- licle transdifferentiation to structures resembling seminifer- For example, the tammar wallaby, a marsupial species, developed ovary-like gonads when treated with estradiol at day 25 postpartum (Coveney et al., 2001). Similarly, male chick embryos treated with estradiol became femi- nized, although this was not permanent and synthetic inhibitors of the estrogen-synthesizing enzyme, P450 aro- matase (P450arom), could induce permanent female- to-male sex reversal (Smith and Sinclair, 2004). Estrogen or inhibitors of P450arom can complete sex reversal in rep- tiles (Bull et al., 1988; Tousignant and Crews, 1994), amphibians (Chardard and Dournon, 1999), and fish (Kobayashi et al., 2003). The effect of androgen treatment been investigated. In this study, we identified the cDNA sequence, including the full-length of the coding region of cholesterol side-chain cleavage enzyme (P450scc), from the leopard gecko; a lizard with temperature-dependent sex determination. Embryonic expression anal- ysis of two steroidogenic enzymes, P450scc and P450 aromatase (P450arom), and four sex steroid hormone receptors, androgen receptor, estrogen receptor a and b, and progesterone receptor, was subsequently conducted. mRNA expression of both steroidogenic enzymes was observed in the brain and gonads prior to the temperature-sensitive period of sex determination. The mRNAs of the four sex steroid hormone receptors were also detected in the brain and gonads at all stages examined. These results suggest the existence of a gonad-inde- pendent sex steroid hormone signaling system in the developing leopard gecko brain. � 2007 Elsevier Inc. All rights reserved. Keys: Reptile; Leopard gecko; Temperature-dependent sex determination; Sex steroid hormone; P450scc 1. Introduction The first sex determination switch is extremely diverse of male gonad-specific genes (Couse et al., 1999). Although gonadogenesis is essentially resistant to exogenous hor- mones in eutherian mammals, exogenous estrogen can Short Com Expression of sex steroid in the embryo of Daisuke Endo, Yoh-Ichiro Department of Biological Sciences, Graduate School of Science, Th Received 28 November 2006; revise Available onli Abstract 0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2007.04.010 unication hormone-related genes e leopard gecko anaho, Min Kyun Park * niversity of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan April 2007; accepted 9 April 2007 1 April 2007 www.elsevier.com/locate/ygcen ology 155 (2008) 70–78 female leopard geckos (15 months of age) were used to identify the partial cDNA of P450scc and examined for tissue distribution of P450scc. They rati steroid hormones in the molecular mechanisms of sex determination or differentiation (Smith and Joss, 1994; Smith et al., 1997; Trant et al., 2001; Akazome et al., 2002). Sex steroid hormones regulate sexually dimorphic traits in other tissues as well as the gonads. Testosterone from the gonads gains access to the brain where it is aromatized to estradiol, which then masculinizes the mammalian brain (McCarthy and Konkle, 2005). In addition, hormonal manipulation affects the formation of the brain structures related to sexual behavior, and can alter traits such as the lordosis of rodents and the song of songbirds (Cooke et al., 1998). Recently, it was suggested that the brain could sexually differentiate independently of gonadal steroids. The gynan- dromorphic zebra finch is genetically male in the right half of the brain and genetically female in the left half. Further- more, the neural song circuit in the right brain has a more masculine phenotype than that of the left (Agate et al., 2003). Since both halves of the brain are exposed to the same circulating hormones, this phenomenon indicates that the genetic sex of the brain cells contributes to sexual differ- entiation. Moreover, both gonadal sex and incubation tem- perature affect male-typical sexual behavior in the leopard gecko (Eublepharis macularius), a lizard with temperature- dependent sex determination (Flores et al., 1994; Rhen and Crews, 1999). This may suggest that incubation tempera- ture directly affects brain organization of the sexual behav- ior of the leopard gecko. Various studies have been conducted to investigate the existence of brain autonomous sexual differentiation (Dewing et al., 2003; Scholz et al., 2006). Extragonadal ste- roidogenesis, for example of neurosteroids, is thought to be one process involved in the regulation of gonad-indepen- dent brain sexual differentiation. Neurosteroids are steroids synthesized in the nervous system either de novo or by the in situ metabolism of precursors from other tissues. They are able to regulate not only adult brain functions but also the sexual differentiation of the brain independently from gonads (Plassart-Schiess and Baulieu, 2001; Holloway and Clayton, 2001). The expression of P450arom in the brain has been reported in many species, including reptiles and birds (Willingham et al., 2000; Trant et al., 2001; Perl- man and Arnold, 2003). However, few studies have focused on early development, or on other genes, such as the gene for cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the first step of steroidogenesis and pro- duces pregnenolone, a major neurosteroid (Nelson et al., 1993). In the present study, the leopard gecko was chosen as an appropriate experimental subject in order to elucidate whether sex steroid hormones can be produced or received in extragonadal tissues during development. There are a number of advantages in using this species. As previously mentioned, it has been suggested that incubation tempera- ture has a direct effect on the leopard gecko brain (Flores D. Endo et al. / General and Compa et al., 1994; Rhen and Crews, 1999). Thus, it is interesting to analyze brain development in this species. Furthermore, were hatched at 29 �C and raised at the same temperature in our labora- tory. Crickets were provided as the main diet three times per week and shelter, water, and a calcium supplement were supplied ad libitum. Ani- mals were maintained on a 14:10 h light/dark cycle at 29 �C. They were anesthetized with sodium pentobarbital (50 mg/kg) and killed by rapid decapitation, followed by complete bleeding. The following tissues were quickly removed, frozen immediately in liquid nitrogen and stored at �70 �C until required: from the female adult leopard gecko, whole brain, pituitary gland, ovary, oviduct, liver, kidney, heart, lung, skin, and adrenal gland; and from the male adult leopard gecko, whole brain, testis, vas def- erens, and kidney. Fertilized eggs of the leopard gecko were acquired less than 24 h after oviposition from the colony maintained in our laboratory and were incu- bated at 26, 32, and 34 �C. In the leopard gecko colony used in this study, sex of almost all embryos incubated at 26 and 34 �C was determined to female and more than 80% of the embryos at 32 �C were to male as described previously (Viets et al., 1993). At 26 �C, six embryos at stage 32 and 33, seven embryos at stage 34 and 35, three embryos at stage 36, 37, and five embryos at stage 38 and 39 were collected from days 8 to 19 of incubation, from 15 to 28, from 28 to 30, and from 30 to 39, respec- tively, according to the criteria of Dufaure and Hubert (1961). At 32 �C, four embryos at stage 32 and 33, six embryos at stage 34 and 35, three embryos at stage 36, 37, and four embryos at stage 38 and 39 were col- lected from days 5 to 9 of incubation, from 12 to 20, at 20, and from 20 to 23, respectively, and at 34 �C, five embryos at stage 32 and 33, six embryos at stage 34 and 35, four embryos at stage 36, 37, and four embryos at stage 38 and 39 were collected from days 6 to 8 of incubation, from 7 to 15, from 15 to 16, and from 18 to 19, respectively. Various tis- sues including brain and gonad–adrenal–mesonephric complex (GAM) past research on reptiles often relied on wild animals and this created problems in obtaining sufficient samples from identical conditions. However, the leopard gecko can be easily maintained and sampled under controlled laboratory conditions, and is also easy to breed. In addition, it reaches sexual maturation within one year, a relatively short time compared with other experimental reptiles, turtles, and crocodiles, which further facilitates research into the devel- opment of the reproductive systems in this species. Another advantage of using the leopard gecko is the availability of molecular information on sex-related genes in the species, such as those for sex steroid hormone receptors (Rhen and Crews, 2001), P450arom (Endo and Park, 2005), GnRH (Ikemoto and Park, 2003; Ikemoto et al., 2004), prolactin (Kato et al., 2005). In the present study, we identified the cDNA sequence of the leopard gecko, including the full-length of P450scc coding region. The expression of two steroidogenic enzymes, P450scc and P450arom, and four sex steroid hor- mone receptors, the androgen receptor, estrogen receptor a and b, and the progesterone receptor, were analyzed in various embryonic tissues. 2. Materials and methods 2.1. Animals All animals were treated according to the guidelines of the Biological Science Committee at the University of Tokyo. Adult fertile male and ve Endocrinology 155 (2008) 70–78 71 were obtained from these embryos. All tissues were frozen immediately in liquid nitrogen and stored at �70 �C until required. 2.2. RNA extraction and cDNA synthesis Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan). The cDNAs used as templates for RT-PCR were synthesized from denatured total RNA using 100 pmol oligo(dT) primer and 100 U of M-MLV reverse transcriptase (Promega, Madison, WI) in a 20 ll reaction volume with incubation at 42 �C for 1.5 h. After incubation, the reactions were diluted to 25 ng/ll and used as templates. The adrenal gland cDNA, used for rapid amplification of cDNA ends (RACE; see Section 2.3), was synthesized from 3 lg of total RNA using a SMART RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer’s instructions. 2.3. Molecular cloning of leopard gecko P450scc cDNA by RT-PCR and RACE RT-PCR was carried out to obtain partial leopard gecko P450scc cDNA from adrenal gland cDNA using degenerate primers, lgscc-dSE01 and lgscc-dAS01, deduced from conserved regions. All the following PCR amplifications were performed in a 20 ll reaction mixture containing each primer at 1 lM, 0.25 u of TaKaRa Ex Taq (TaKaRa, Shiga, Japan), each dNTP, and Ex Taq buffer (TaKaRa). The reaction conditions for RT-PCR were as follows: 94 �C for 5 min, 35 cycles of 94 �C for 40 s, 58 �C for 30 s, 72 �C for 1 min, and 72 �C for 7 min. The amplified prod- ucts were separated by electrophoresis in 1.5% agarose gel and visualized using ethidium bromide staining. DNA fragments were extracted using a QIA Quick Gel Extraction Kit (Qiagen K.K., Tokyo, Japan) and directly sequenced using a dRhodamine terminator cycle sequencing FS Ready Reaction Kit (Applied Biosystems, Tokyo) by primers lgscc-dSE01 and lgscc-dAS01. After sequencing, sense and antisense gene-specific primers, lgscc-SE01, lgscc-SE02, and lgscc-AS01 were designed based on the sequences. Using these primers, the inner part of the cDNA fragments amplified by the primers, lgscc-dSE01 and lgscc-dAS01, were sequenced. After determining the partial sequence of P450scc cDNA, sense and anti- sense gene-specific primers were designed based on the sequence (Table 1). RACE was carried out to obtain the complete sequence. 3 0- and 5 0-RACE was performed with SE03 and the Nested Universal Primer (NUP) of the kit (see Section 2.2), and NUP and AS02, respectively. Each PCR condi- tion was as follows: 94 �C for 5 min, 35 cycles of 94 �C for 40 s, 64 �C 30 s, 72 �C for 2 min, and 72 �C 7 min. The amplified products were sequenced as described above. 2.4. Comparison of the amino acid sequences of various P450scc The CLUSTAL X program (version 1.81) was downloaded from ftp:// ftp-igbmc.u-strasbg.fr/pub/ClustalX/ and used with default settings to align the deduced amino acid sequences of P450scc of the leopard gecko and other species in relation to each other (Thompson et al., 1997). The amino acid identity was calculated using GeneDoc software (version 2.6.002) (Nicholas and Nicholas, 1997). The GenBank accession numbers of P450scc used in the comparisons and phylogenic analysis are as follows: human NM000781; mouse NM019779; American alligator DQ007995; chicken NM0010017; zebra finch AY633556, and zebra fish NM152953. 2.5. Molecular phylogenic analysis The amino acid sequences of the entire ORFs of P450scc in the leopard gecko and several species from all vertebrate classes were aligned using CLUSTAL X with default settings. The alignment of the amino acid sequences was used to generate the phylogenic tree, using the neighbor- joining method (Saitou and Nei, 1987). Bootstrap values were calculated with 1000 replications to estimate the robustness of internal branches. sequ ea A CC G TTAGGGCTCCTGCAGG-3 For sequencing and RT-PCR GC 0 CA CA TG CC GG G AC GG lgscc-AS05 5 -CAGTTTCTCCCT lgscc-AS06 5 0-GGGAATTCCTGG GAAAAGTGTCCCCCAC -3 For RT-PCR GA 0 G TC AG AG GG G AC AA 72 D. Endo et al. / General and Comparative Endocrinology 155 (2008) 70–78 lgP450arom lgarom-SE01 5 0- CAATTTTGA lgarom-AS01 5 0-GGATGGGAT lgERa lgERa-SE01 5 0-GATTCGGAAA lgERa-AS01 5 0-TGGCTCGGCA lgERb lgERb-SE01 5 0-GTGGAACAC lgERb-AS01 5 0-GGGCTTGTGC lgAR lgAR-SE01 5 0-ATGAAGCAG lgAR-AS01 5 0-GCAGGTTACG lgPR lgPR-SE01 5 0-CTGGCATGGT lgPR-AS01 5 0-GACTACACAC Table 1 Oligonucleotide primers used for degenerate PCR, RT-PCR, RACE, and Gene Name Nucleotide sequenc lgP450scc lgscc-dSE01 5 0-ATHTACAGGG lgscc-dAS01 5 0-GGNTCWCGR lgscc-SE01 5 0-GTGAAGCTTG lgscc-SE02 5 0-GGGACACGC lgscc-SE03 5 0-CAGAACTGCT lgscc-SE04 5 0-TCAAGGAGA lgscc-SE05 5 0-GAATTTATGC lgscc-SE06 5 0-CATCTTTTTC lgscc-SE07 5 0-CCTTGGCTAA lgscc-AS01 5 0-CTCAGCAACG lgscc-AS02 5 0-ATTCAGGAGA lgscc-AS03 5 0-GCATGTAGAC lgscc-AS04 5 0-GTGTAAATTG 0 N represents all four nucleotides. a Abbreviations for degenerate nucleotides: Y, C or T; R, G or A; K, G or CAGTTTTTCGTTGG -3 For RT-PCR ACCGCAGAGGTGG-3 0 For RT-PCR TAGCAAGGCACTG-30 For RT-PCR TCCAATCTATCCC-3 0 For RT-PCR TCACTGCCGCTG-3 0 For RT-PCR ATGACCCTTGGAGC-3 0 For RT-PCR AATCCTGGTAAGGC-30 For RT-PCR TTGGGGGTCGAAAG-30 For RT-PCR AGAAGCTGCCTCTC-3 0 For RT-PCR AAAAGGTGATATG-3 For RACE and sequencing ATAAGGCTCCACCC-30 For sequencing TGGGTCGGGACCC-3 0 For sequencing ATCCATATGCTCG-3 0 For sequencing ATGGGATTCAAG-3 0 For sequencing ACCGATTTCAGCAC-30 For sequencing and RT-PCR GCACAAATTTGTCC-3 0 For RACE and sequencing GCTTGACAAAGTC-3 0 For sequencing CCAAATTTCTGG-3 0 For sequencing GTAAATTGGCCCA-3 0 For sequencing AGATTTGTAGG-30 For sequencing 0 encing Usage GAARNTNGG-30 For degenerate PCR CATRGCRTA-30 For degenerate PCR CGCCATGACCGCCTG-30 For sequencing 0 T; H, A, T or C; W, A or T. The GenBank accession numbers of P450scc used in the comparisons and phylogenic analysis are as follows: chacma baboon AY702067; Norway rat BC089100; golden hamster AF323965; cow NM_176644; pig NM_214427; horse AF031664; goat D50058; sheep D50057; channel cat- fish AF063836; rainbow trout S57305. 2.6. Expression analysis RT-PCR was performed to identify the possible source of sex steroid hormones in the adult and developing leopard gecko. Twenty-five nano- grams of cDNA from various organs of adult and developing leopard geckos were amplified using specific primer sets (Table 1). b-Actin was used as an internal control of cDNAs. The PCR conditions were as follows: 94 �C for 5 min, 35 cycles (30 cycles for b-actin) of 94 �C for 40 s, 64 �C for 30 s, 72 �C for 1 min, and 72 �C for 7 min. The amplified products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. No band was detected using total RNA without reverse transcription under PCR conditions with the primer sets for this expressional analysis. The specificity of PCR was confirmed by sequence analysis. The GenBank accession numbers of genes used in the expression analysis are as follows: 450scc AB252075; P450arom AB18592; ERa AB240528; ERb AB240529. The sequences of leopard gecko androgen receptor and progesterone receptor have not been deposited in GenBank. The sequences of the leopard gecko androgen receptor and progesterone receptor were therefore taken from the report of Rhen and Crews (2001) and Endo and Park (2003), respectively. : : : : : : : * 20 * 40 * 60 * 80 * -----MGFTPSLG-----------RCTFVS-------------SRESSQAIQRIAGQLEKQWLNLYRFWQEDGFRNVHNIMVHRFQKFGP MLSRAAPIAG.FQ-----------A.RCAGGIPALAGVHYPLP.SSGARPFDQ.P..WRAG.....H..K.G..H......AS....... MLARV.TKPGA.R-----------G.PRGAAARCRRLGGAGGAVPSAPRPFNQ.P..WRAG........R.G.LSA..LS.AQ..R.... MLAKG.PPRSV.VKG--YQTFLSAPREG.GRLRVPTGEGAGIS-TR.PRPFNE.PSPGDNG.....H..R.T.THK..LHH.QN...... MLAKG.SLRSV.VKG--CQPFLSPTWQG---PVLSTGKGAGTS-TS.PRSFNE.PSPGDNG.....H..R.S.TQK..YHQ.QS...... -MARWNVTFAR.D--------QSLSSLKNLLQVKVTRSGRAPQNS-.V.PFN..P.RWRNSL.S.LA.TKMG.L....R....N.KT... : 61 : - : 79 : 79 : 87 : 84 : 80 : : : : : : : 100 * 120 * 140 * 160 * 180 IYREKLGNYESVNSIDPGRRCTVFNTEGLYPERFSVPSWMAYRDFRNKPYGVLLKKGEAWRHDRLTLNKEVLSPWAMDKFVPLLNEVGQD ..I...EDAA...K...........SP......H...S......N.....F...I........QV..N............ .......V.....I.S.RDAA...K....L.......P.................T.....S............QV..S........S.. .......VH....I.S..DAA...QA..AL....R..P.................T.....S...L..Q.A.A.A..AA.....SA.... ........V...YV...EDVAL..K...PN....L..P....HQ.YQ..I......SA...K...A..Q...A.E.TKN......A.SR. .......TL...YI...KDASI..SC..PN....L..P....HQ.YQ..I...F.SSD...K...V..Q...A.G..KN.....EG.A.. .......I.D..YI.K.EDGAI..KA..HH.N.IN.DA.T.......QK......E.K...T...I......L.KLQGT.....E..... : 151 : 80 : 169 : 169 : 177 : 174 : 170 : : : : : : : * 200 * 220 * 240 * 260 * FVKRVYMQIERSQQGRWTADLTNELFRFALESVSNVLYGTRLGLLQDIIDPDAQQFINAITTMFHTTTPMLYIPPDFLRRISSKTWQDHI ....R......GR......F............CH....E.......F...E..R......M............VKLFHW.N........K ....ARA...Q.GRE.....F.H..........CH....E.......F...E.........L.............AL..H.N....R... ....ARA.ARH.GH.C..G.F.H..........CH....Q.......F.Q.E..R..E..AR.....A.......AL....R....RE.. ..SV.HRR.K.AGS.N..G....D.....F.........E.Q....E....E..R.....YQ.....V...N....LF.LFR....K... ...V.HRR.KQQNS.N..GV...D....SF....S....E......E....